Effect Of Leptin On Bovine Oocyte In Vitro Maturation

With the continuous development of animal embryo transfer technology,the demand for high-quality oocytes has become a major topic of discussion.Leptin is a multifunctional hormone mainly secreted by adipocytes,which can not only regulate the metabolism of the body,but also directly act on gonadotropic axis and ovary,and participate in the regulation of puberty and reproductive development.The purpose of this study was to explore the role of leptin in bovine oocyte maturation in vitro and its potential mechanism.In this study,firstly,the cumulus expansion,meiotic process and cell division were studied by adding 0,1,10 and 20 ng/ml leptin to the in vitro maturation(IVM)medium,and the optimal leptin concentration was obtained.Then,the effect of leptin on reactive oxygen species(ROS),glutathione(GSH),mitochondrial membrane potential(MMP)and cathepsin B(CB)were studied by adding the optimal leptin concentration to IVM medium,and real-time fluorescence quantitative PCR(q RT-PCR)was used to analyze expression of antioxidant related genes SOD1,SIRT1,GPX4 and apoptosis related genes Survivin,Caspase-3,Bax.The results showed that Leptin-treated had no significant effect on cumulus cell expansion(P>0.05),but 10 ng/ml Leptin-treated could accelerate meiosis process.Compared with the control group,1 ng/ml and 20 ng/ml leptin groups,it significantly increased the proportion of oocytes developing to MⅡ(81.67±3.51%vs.64.00±2.01%,67.33±2.51%,67.67±4.04%;P<0.05).After in vitro fertilization,the proportion of oosperm reaching 2 cells in10 ng/ml leptin treatment group was significantly higher than that of the control group,1ng/ml and 20 ng/ml leptin groups(67.67±2.51% vs.56.00±2.00%,58.67±2.08%,52.00±2.00%;P<0.05).The division rate of 8 cells treated with 10 ng/ml leptin was significantly higher than that of the control group,1 ng/ml and 20 ng/ml leptin groups(44.67±1.52%vs.30.33±2.07%,31.67±2.08%,26.01±1.05%;P<0.05).The results showed that the optimal concentration of leptin was 10 ng/ml.Further study showed that 10 ng/ml leptin can significantly decrease the level of ROS and increased the level of GSH(P<0.05),and up-regulated the expression of antioxidant related genes SOD1,SIRT1 and GPX4(P<0.05),compared with the control group.In addition,10 ng/ml leptin also significantly increased the level of MMP(P<0.05).At the same time,10 ng/ml leptin could significantly reduce the activity of CB(P<0.05),accompanied by a significant up-regulation of survivin expression about antiapoptotic-related genes and a significant down-regulation of Caspase-3 and Bax expression about apoptotic-related genes(P<0.05).In conclusion,the results indicated that during in vitro maturation,leptin(10 ng/ml)can reduce oxidative stress and apoptosis,improve MMP to promote oocyte maturation.