Effect Of Methomyl On Oocytes And Development Of Mouse Preimplantation Embryos In Vitro

With the growth of agricultural economy,the emergence of pesticides has provided a great relief to people’s lives.But,the high use of pesticides also poses a potential threat to human and animal habitats.Methomyl(MET)is a urethane wide-spectrum fungicide that is extensively used on vegetation,grain,fruit,cotton and other crops.Some studies have found that MET is an endocrine disruptor.The World Wildlife Fund listed MET as environmental estrogen(EE)in 1997.MET has been proven to cause various toxicity in animals and to affect human health as well.At present,there are many studies on MET,but there are few reports on the effects of MET on oocytes and early embryo development in female mammals.The objective of this work is to examine the toxic impact of MET on oocytes and early embryonic development from in vitro using mice as a test animal model,thus giving some theoretical basis for mammalian reproductive diseases.This study is mainly subdivided into two parts as follows:Part Ⅰ: Effect of MET on oocyte maturation in miceThis report discussed the affect of MET on mouse oocytes ripening and the potential structure.First,the sensitivity of mouse oocytes to MET gradient concentrations was explored,and the first polar body efflux rate was used as a marker.The results found that the first polar body expulsion rate was reduced significantly after 60 and 100 μM MET treatment,and all oocytes were lethal after 200 μM MET treatment.To explore further the effect of MET on oocyte maturation,the present study also tested the oxidative stress,spindle morphology and mitochondrial function in mouse oocytes using immunofluorescence staining,and the expression of apoptosis-related genes using q RT-PCR.The results showed that the oocyte ROS levels were significantly increased in the 60 μM MET group.After MET treatment,the MI stage mouse oocytes showed abnormal spindle morphology.At the same time,the mitochondrial membrane potential of oocytes in the MET group was significantly reduced,resulting in a decline in the maturation quality of mouse oocytes.In addition,MET can also induce apoptosis,as evidenced by raised mRNA expression of the pro-apoptotic genes Bax and Caspase-3.Therefore,our results suggest that MET can cause oxidative stress and apoptosis in oocytes to interfere with the maturation of mouse oocytes.Part Ⅱ: Effect of methomyl on early embryonic development of mice in vitroStudies have shown that EE can have toxic effects on early embryos even at very low concentrations,and even cause fatal damage to the subsequent development of embryos.In the proposed research,the effect of MET on early mouse embryonic development in vitro and its mechanism were studied.First,the early mouse embryos were cultured in vitro for 24 h or 96 h by drug MET treatment,and the 2-cell rate and blastocyst rate of early embryos were counted by morphological observation.Using immunofluorescence staining and q RT-PCR,the total blastocyst cell number and apoptosis rate,reactive oxygen species level,mitochondrial membrane potential,LC3 protein expression,glutathione content,γ-H2 A.X expression level and the expression of antioxidant-related genes were detected in early embryos.It was noted that there was no significant difference in the2-cell rate of early embryos in different MET concentration groups compared with the control group,but the addition of 30 μM MET could significantly reduce the blastocyst rate.MET raises oxidative stress and disrupts mitochondrial function,which may account for the reduced blastocyst rate.In addition,this study found that MET treatment can promote the apoptosis of blastocysts and reduce the total number of blastocysts.The level of autophagy and the degree of DNA damage in early embryos were significantly higher after MET treatment,and the mRNA transcript levels of antioxidant-related genes Sod2,Cat and Gpx3 were significantly lower.The results suggest that MET treatment reduces the ability of early embryos to develop in vitro and exerts its toxic effects by causing oxidative stress and disrupting mitochondrial function.In summary,this work showed that MET has toxic effects on oocyte maturation and early embryonic development in mice,as evidenced by reduced first pole body expulsion rate and blastocyst rate.MET caused oxidative stress,abnormal spindle morphology,and inhibited oocyte maturation;MET also inhibited autophagy and DNA damage,which reduced the developmental capacity of early embryos.