The Effects Of Aristolochic Acid On The In Vitro Maturation And Fertilization Of Porcine Oocytes

The quality of mammalian oocytes is one of the prerequisites that determine the animal fertility.The declining oocyte quality would cause the oocyte maturation arrest,ferilization failure and embryonic development,severely limiting the animal reproductive performance and animal husbandry development.The factors influencing the oocyte quality include genetic and environmental aspects,implicated in the key biological events during oocyte maturation such as cytoskeleton organization,chromosome segregation,mitochondrial dynamics and cortical granule distribution.Increasing evidence reveals that environmental factors extensively affect the oocyte quality and animal fertility compared to genetic factors.Air,water and soil pollutants as well as feed ingredients and feeding management level to a certain extent perturb the livestock growth and reproductive performance.Although some environmental factors have been reported to impair the oocyte quality,more factors are not known.Therefore,the study regarding whether these potential toxicants in the environment will affect oocyte quality and its possible mechanism would extend our understanding about the biological features of oocyte maturtation and fertilization disrupted by the environmental factors,and provide new ideas for improvement of animal oocyte quality and reproductive performance.Taking advantage of porcine oocytes as the model,as well as oocyte in vitro maturation,in vitro fertilization,embryonic development systems and immunofluorescence,immunoblotting methods,the present study investigated the effects of AAI(aristolochic acid I)on the in vitro maturation and fertilization of porcine oocytes and revealed their potential mechanisms.The present study is divided into three parts,and the experimental content as well as the research results are as follows:Experiment 1 AAI disrupts the nuclear maturation of porcine oocytesAAI is a compound derived from various herbaceous plants of the genus Aristolochia and Asarum.It was later discovered that AAI is nephrotoxic as well as carcinogenic and can cause kidney failure and liver cancer.In addition,AAI is a soil pollutant which can be absorbed by soil and accumulated in a time and dose-dependent manner in the food crops,thereby entrying into the food chains of humans and animals to induce the diseases.It has been reported that AAI is also a harmful ovarian toxicant,which can significantly reduce the the ovarian reserves and the number of ovulated oocytes.However,it is not known whether AAI affects the oocyte quality.Therefore,the present study was conducted to investigate the effects of AAI on the porcine oocyte meiotic maturation and fertilization ability.The results showed that AAI considerably inhibited the expasion of cumulus cells and reduced teh polar body extrusion rate of porcine oocytes at a concentration of 100 μM,indicating that AAI hindered the in vitro maturation of oocytes.Given that the process of oocyte maturation is divided into nuclear maturation and cytoplasmic maturation,and the chromosome contains a large amount of genetic information which regulates the cell development,we thus tested the spindle assembly and chromosome alignment to examine the impact of AAI on the nuclear maturation of oocytes.The results revealed that AAI treatment led to the disorganized spindle assembly by showing the multipolar or distorted morphologies,and the scattered chromosomes that disorderly aligned at the equatorial plate.The spindle assembly abnormality is usually related to the microtubule dynamics;therefore the stability of microtubule was further studied.Both immunofluorescence and immunoblotting analyses manifested that AAI treatment remarkably decreased the acetylation level of α-tubulin.Collectively,our data indicate that AAI treatment compromises the nuclear maturation of porcine oocytes.Experiment 2 AAI perturbs the cytoplasmic maturation of porcine oocytesNuclear maturation is not always synchronized with the cytoplasmic maturation during oocyte maturation,and thus the completion of nuclear maturation does not ensure the normal progress of cytoplasmic maturation.Therefore,we further investigated the influence of AAI treatment on the cytoplasmic maturation by evaluating the cytoskeleton and organelles in the cytoplasm.Actin,one of the key cytoskeletons,plays a vital role in the spindle positioning and cortical polarization during oocyte meiotic maturation.Immunostaining and fluorescence intensity measurement showed that AAI treatment dramatically impaired the accumulation of actin filaments on the plasma membrane of oocytes.In addition,the normal distribution of mitochondria and cortical granules is regarded as the important indicator of oocyte cytoplasmic maturation.We found that AAI treatment markedly increased the rate of mis-located mitochondria and reduced the mitochondrial signals,suggesting that mitochondrial integrity is compromised.Furthermore,the distribution of cortical granules was also disrupted by displaying the discontinued or completely absent localization patterns in the subcortical region of oocytes.Ovastacin is a pioneer component of cortical granules in mammals that is required for the post-fertilization cleavage of sperm binding site in the zona pellucida to prevent polyspermy.If the distribution of ovastacin is ont correct,it will lead to the fertilization failure.Immunofluorescence results indicated that the localization of ovastacin was abnormal and its signals were faded in the subcortical region of oocytes,which might be caused by the premature exocytosis of ovastacin,suggesting the disturbed sperm binding ability of AAI-treated oocytes.Together,AAI treatment impairs the cytoplasmic maturation of porcine oocytes by compromising actin organization,mitochondrial integrity,cortical granule distribution and ovastacin exocytosis.Experiment 3 AAI impairs porcine oocyte maturation and fertilization by inducing oxidative stress to cause DNA damage and apoptosisThe abnormal localization of ovastacin predicts that AAI treatment might affect the fertilization process of porcine oocytes.To this end,we tested the fertilization potential of oocytes by sperm-oocyte binding assay and in vitro fertilization experiment.The results showed that AAI treatment significantly reduced the number of sperm binding to the zona pellucida of oocytes and the rate of 2-cell embryos,which is consistent with the observation that ovastacin was prematurely released to cleave the sperm binding sites and thereby resulting in the defective sperm binding and fertilization.As a cellular toxicant,AAI can induce the oxidative stress to cause DNA damage in somatic cells,and hence disrupting the normal physiological activities of cells and accelerating the apoptosis.To test whether this is the case in the oocytes,we assessed the ROS level,DNA damage level and apoptotic rate.The data manifested that AAI treatment prominently elevated the ROS level,accumulated DNA damage and increased the apoptotic rate.In summary,our findings demonstrate that AAI adversely influences the porcine oocyte maturation and fertilization via the excessive ROS-induced DNA damage and apoptosis.