Toxicological Studies On The Environmental Estrogens To Mouse Oocytes And The Corresponding Protective Methods

Reproductive and developmental dysfunction is one of the major public health problems that seriously affect human health.Exogenous factors such as disease,stress and chemical substances interfere hypothalamus-pituitary-gonadal axis,leading altered ovarian cycle,decreased fertility,offspring dysplasia and spontaneous abortion.In recent years,exposure to environmental estrogens(EEs)is considered as a great risk factor affecting women's reproductive function,in addition to aging,diabetes and smoking.A large number of studies have reported that EEs interfere with the endocrine system,break the hormone balance,destroy ovarian and uterine normal physiological activities,and ultimately cause adverse reproductive results.However,as a basic part of the female reproductive process,whether EEs would disturb oocyte meiotic maturation and the related mechanisms have not been reported.In this paper,we selected methoxychlor and mancozeb as representative to study their effects on oocytes,also with an attempt to find the corresponding protective methods.Methoxychlor(MXC)is an organochlorine pesticide that has been widely usedas a substitute for pesticide control in the world since the ban of DDT occurred in the 1970s.Previous studies linked MXC exposure with hyperestrogenism and other reproductive disorders,which reflect its ability to suppress the activity of steroidogenic enzymes.In female mice,in utero developmental exposure to MXC through their mother led to irregular estrous cycles,decreased progesterone levels,reduced litter sizes,and more frequent delivery of dead fetuses.In this study,we used in vitro maturation(IVM)model to investigate the effect of MXC on oocyte meiosis.Our results showed that maturation rates of MXC-treated oocytes were lower than those of controls,which was due to abnormal spindle morphologies,dysfunctioned microtubule organized centers(MTOCs)and activated spindle assembly checkpoints BubRl and Mad2.In addition,MXC increased the level of reactive oxygen species(ROS)in mouse oocytes,with the evidence of increased fluorescence intensity of ROS-labeled probe 2',7'-dichlorofluorescin diacetate.We also observed the ROS-mediated cytotoxicity occurs in MXC-treated mouse oocytes,such as DNA double-strand breaks and lipid peroxidation.Finally,we found the distribution of mitochondria was changed in mouse oocytes under MXC conditions.In conclusion,these data suggested that MXC reduces the developmental potential of mouse oocytes,and this was achieved by impairing cellular ROS metabolism.Mancozeb,a mixture of ethylene-bis-dithiocarbamate manganese and zinc salts,is one of the most widely used fungicides in agriculture.Mancozeb could lead to mitochondria dysfunction,cellular anti-oxidation enzymes depletion and apoptotic pathways activation.Previous studies indicated the exposure of mancozeb through mother would lead to irregular estrous cycles,decreased progesterone levels,reduced litter sizes,and more frequent delivery of dead fetuses.In this study,we attempt to relieve mancozeb-induced female reproductive toxicity with resveratrol,a natural product with estrogen and anti-apoptotic effects.We divided the mice into four groups:negative control group,positive control(mancozeb)group,low concentration of resveratrol(100 mg/L)group and high concentration of resveratrol(200 mg/L)group.After 4 weeks,we evaluated the reproductive behivors of each group with the status of offspring,ovaries and oocytes.Results showed that resveratrol could alleviate mancozeb's adverse effects,such as declined fertility,decreased ovary weight and primary follicles.Besides,mancozeb treated oocytes displayed suboptimal developmental competence and this can also be improved by treatment of resveratrol.More detailed investigation of these processes revealed that mancozeb increased reactive oxygen species,causing cell apoptosis and abnormal epigenetic modifications,and resveratrol can block these cytotoxic changes.Collectively,our results showed that resveratrol can alleviate mancozeb induced infertility and this was mainly through the correction of apoptotic tendency and the abnormity of cellular epigenetic modification.