Mechanism Study Of Ethylene Glycol Butyl Ether Impairment On The Quality Of Mammalian Oocytes

In recent years,the impact of environmental pollution on animal growth and development has been widely concerned.Environmental pollutants can invade the body through gastrointestinal tract,respiratory tract,and skin contact,and damage the health of animals and humans in the form of acute,cumulative or chronic hazards.Environmental pollutants entering the body damage a variety of organs and tissue cells by interfering with the synthesis and release of hormones,receptor recognition and binding,resistance and immune response,and thereby affecting the animal health.In addition,environmental pollutants destroy oogenesis and spermatogenesis by impairing ovarian and testicular development to damage the normal function of the reproductive systems,and thus affect the reproductive performance of livestock.Ethylene glycol butyl ether(EGBE),a type of glycol ethers,is a common chemical used in both industrial and household products.Increasing animal studies have indicated that EGBE not only has negative effects on tissues and organs,but also produces reproductive problems,such as testicular damage,decreased female fertility,death of embryos and birth defects.However,the impact of EGBE on female germ cells and its potential mechanism are still unclear.Taking advantage of mouse and porcine oocytes as the model,we explored the adverse effects of EGBE on the oocyte meiotic maturation and their potential mechanisms by gavage in mice and supplementation in the culture medium during porcine oocyte in vitro maturation.The findings provide a theoretical basis for revealing the impact of environmental pollutants on the reproductive health of animals and humans,and provide ideas for improving oocyte quality and reproductive performance of animals.The study is divided into two parts,and the specific experimental content as well as results are shown as follows:Experiment 1 EGBE deteriorates mouse oocyte quality via impairing mitochondrial functionWe administered mice with different doses of EGBE(100 mg/kg/day,250mg/kg/day,300 mg/kg/day)for 10 days by gavage to observe the effects of EGBE on the meiotic maturation and fertilization of mouse oocytes.By assessing the in vivo maturation rate of EGBE-exposed oocytes,we selected 300 mg/kg/day of EGBE for subsequent studies.Meanwhile,we found that EGBE exposure at a dose of 300mg/kg/day had no effect on the meiotic resumption(germinal vesicle breakdown,GVBD),but significantly reduced the rate of first polar body extrusion(PBE)during in vitro maturation of oocytes.Further investigation of oocyte maturation-related events and core regulatory factors found that EGBE exposure disrupted the cytoskeleton dynamics to cause the defects in oocyte maturation,by showing aberrant spindle assembly,incorrect chromosome alignment and abnormal actin polymerization.In addition,EGBE exposure impaired the distribution of cortical granules and release of ovastacin in mouse oocytes,resulting in the failure of sperm binding and fertilization.Single-cell transcriptome sequencing analysis revealed that EGBE-induced oocyte meiotic defects were caused by mitochondrial dysfunction.EGBE exposure compromised the distribution and function of mitochondria,leading to the production of excessive reactive oxygen species(ROS),accumulation of DNA damage,and occurrence of apoptosis.Collectively,our study demonstrates that impaired mitochondrial function is the main cause for the decline of mouse oocyte quality induced by EGBE exposure.Experiment 2 EGBE destroys the cytoskeleton dynamics to cause the maturation defects in porince oocytesDuring in vitro maturation of porcine oocytes,different concentrations of EGBE(50,100 or 200 μM)were added to the maturation medium to evaluate the two key indicators of porcine oocyte maturation,the rate of the first polar body extrusion and the extent of cumulus cell expansion.Based on the result of polar body extrusion rate,we selected 100 μM EGBE for subsequent studies.Quantitative data showed that EGBE treatment at a concentration of 100 μM considerably decreased the first polar body extrusion rate and inhibited the expansion of cumulus cells of porcine oocytes,indicating that EGBE hinders the nuclear maturation of porcine oocytes.As the damage to cytoskeleton assembly usually leads to meiotic defects,we examined the cytoskeleton dynamics.The data showed that EGBE treatment disrupted spindle assembly by weakening the stability of microtubules,accompanied by the misalignment of chromosomes.In addition,EGBE treatment also impaired the integrity of actin filaments,another basic component of the cytoskeleton in oocytes.Therefore,these findings indicate that EGBE induces the dynamic defects of the cytoskeleton assembly during porcine oocyte maturation.Mitochondrion is the main site for ATP production in oocytes,and its distribution pattern has been regarded as one of the key indicators for evaluating oocyte cytoplasmic maturation.Our findings have found that EGBE treatment interfered with the mitochondrial localization,mitochondrial membrane potential and ATP production,indicating that EGBE compromises the distribution and function of mitochondria,thereby impairing the cytoplasmic maturation of porcine oocytes.In addition,EGBE treatment increased levels of ROS and induced the accumulation of DNA damage as well as the occurrence of apoptosis.In summary,EGBE deteriorates oocyte quality by impairing mitochondrial function and cytoskeleton assembly,and induces the generation of oxidative stress and apoptosis in oocytes.The adverve impact of EGBE on oocyte quality is conserved between mice and pigs.