The Toxic Effects And Possible Mechanisms Of Bisphenol A And DEHP On Porcine Oocyte Maturation In Vitro

With the development of economy,many pollutants have a a potential threat to human and animal’s living environment in the modem chemical industry.Such as bisphenol A(BPA)and di-(2-ethylhexyl)phthalate(DEHP),they are widely used in the plastics industry,such as the cups,baby bottles,lunch boxes,packaging bags,toys and so on.Some studies found that BPA and DEHP are endocrine disorders,they disturbed the endocrine system,and they are linked to several diseases,such as infertility and so on.In the research of DEHP,some related experiments also studied the metabolic pathway in vivo,including the main active intermediate mono(2-ethyhexyl)phthalate(MEHP)and 2-ethylhexanol,The metabolites of MEHP also may cause some toxic effects on human body.BPA and DEHP were found to be endocrine disrupting chemical(EDCs),therefore,the research that the toxic effects and possible mechanisms of BPA and MEHP on porcine oocyte maturation in vitro,the experiment can provide theoretical basis for clinical diseases.We make the porcine as the experimental model in this experiment,the methods including using in vitro culture,drug treatment,parthenogenetic activation,immunofluorescence staining,reactive oxygen species generation determination,annexin staining(annexin-V)and fluorescence quantitative PCR(real time PCR)and Western blot(Western blot)and so on.Firstly,by the drug treatment to observe the expansion of peripheral layers cumulus in porcine oocytes maturation and the rate of first polar body(pbI),which we can judge the influence of EDCs.Subsequently,the cell cycle were been statistical for 44 h and 60 h cultured,the aim is to check the cell cycle progression by laser confocal microscopy.Observation by fluorescence staining,whether endocrine disruptors affect the microtubule assembly or not,by Image J to determine the fluorescence intensity of the microfilament and to judge whether the actin filaments is affected.The localization and expression level of p-MAPK and LC3 were assessed by Western blot.EDCs can change the levels of histone methylation and DNA methylation by immunofluorescence staining,at the same time the trend of gene be checked by real time PCR.Some studies have found that oxidative stress can cause apoptosis and autophagy effect,so we detected the expression level of ROS and the levels of early apoptosis and autophagy.Till now there is still little research focused on EDCs influence on porcine oocyte maturation and especially the mechanisms of its toxicity.Collectively,our studies checked the toxic of BPA,DEHP and its active metabolite MEHP on porcine oocyte maturation in vitro.This study is divided into two parts,the main results are as follows:Experiment 1.The toxic effects and possible mechanisms of Bisphenol A on porcine oocyte maturation in vitro.The experimental results are as follows:1.The rates of oocytes maturation significantly decreased with BPA exposure in vitro,the reason might be due to the delayed cell cycle progression on porcine oocyte maturation in vitro.2.Compared with the controls,BPA treatment resulted in abnormal cytoskeletons on porcine oocytes in vitro,such as chromosome alignment,spindle morphology and aberrant actin distribution.The results were further confirmed by the reduced p-MAPK level.3.By immune fluorescence analysis,the results showed that the levels of histone methylation(H3K4me2)and DNA methylation(5mC)were changed after BPA exposure,thereby the results indicating that the epigenetic modification were disturbed after BPA exposure.4.We also found that the higher rates of early stage apoptosis/autophagy after BPA exposure.The reason might be due to the increased level of oxidative stress.The localization and expression level of LC3 were assessed by Western blot.To sum up,our results showed that porcine oocytes maturation were disrupted after BPA exposure,through disrupting cytoskeletal dynamics,epigenetic modifications and apoptosis/autophagy to check their toxic.Experiment 2.The toxic effects and possible mechanisms of di-(2-ethylhexyl)phthalate and its metabolite mono(2-ethylhexyl)phthalate on porcine oocyte maturation in vitro.The results are as follows:1.DEHP treatment did not effects the expanision of granule cell on porcine oocytes maturation,and it can not affect the rate of oocyte maturation.2.MEHP is an active metabolite of DEHP,the results showed that the granule cell diffusion of COCs were weakened after MEHP treatment,and the rates of porcine oocytes maturation were significantly decreased after MEHP exposure in vitro.The reason might be due to the delayed cell cycle progression after MEHP treatment.3.Compared to the controls,the rates of abnormal cytoskeletons were significantly increased after MEHP treatment on porcine oocytes in vitro,such as aberrant actin distribution,spindle morphology and chromosome alignment.4.The levels of histone methylation(H3K4me2)and DNA methylation(5mC)were altered after MEHP treatment by immune fluorescence intensity analysis.The results showed that MEHP treatment can changed the epigenetic modification.5.MEHP treatment can induce the early apoptosis and autophagy,this may be due to the changed levels of oxidative stress.In order to verify the results,the level of LC3 localization and expression were significantly increased by Western blot.Finally,our experimental results showed that MEHP exposure can affect the rates of porcine oocyte maturation in vitro.The results can be proved by cytoskeletal dynamics,early apoptosis/autophagy and epigenetic modifications.