| In vitro maturation of bovine oocytes is a key step before bovine embryo transfer,but in vitro environmental factors such as oxidative stress inhibit maturation of bovine oocytes.Due to the loss of the body’s own defense system,excessive reactive oxygen species(ROS)can adversely affect the maturation of oocytes in vitro.The quality of oocyte maturation is directly related to the development of subsequent embryos.In the process of embryonic development,often accompanied by a "block",the phenomena of oxidative stress are thought to be one of the important cause of embryo development block,if an imbalance in the body redox system,reactive oxygen species(ROS)concentration beyond the removal ability of cells,can cause lipid peroxidation and cell signaling protein oxidative modification,DNA chain rupture,etc.,This can have serious consequences on oocytes and embryos.Limonin(Lim),a highly oxidized tetracyclic triterpenoid,is the main source of bitterness in citrus fruits.Because of its anti-tumor activity,antibacterial,antiviral,neuroprotective,antioxidant activity and other functions,more and more researchers pay attention to.However,the effect of limonin on mammalian reproduction has not been reported.In this study,bovine oocytes were used as research object and limonin was added to bovine oocyte in vitro maturation culture system for the first time,aiming to explore the effect of limonin on bovine oocyte maturation in vitro and provide theoretical basis for optimizing the in vitro culture system of bovine oocytes and improving the efficiency of embryo transfer.Experiment 1: Effect of limonin on in vitro maturation of bovine oocytesThe rate of oocyte development to MII stage in 20 μM/L Lim treatment group was significantly higher than that in the other three groups(P<0.05)after Lim treatment at different concentrations(0 μM/L,10 μM/L,20 μM/L and 50 μM/L).The ratio of 50 μM/L Lim treatment group to MII stage was significantly lower than that of the other three groups(P<0.05).The 8-cell rate of the control group was not significantly different from that of the 10 μM/L Lim group(P>0.05),but was significantly lower than that of the 20 μM/L Lim group(P<0.05).The blastocyst rate in the control group was not significantly different from that in the 10 μM/L Lim treatment group(P>0.05),but significantly lower than that in the 20 μM/L Lim treatment group(P<0.05).In addition,ROS levels and glutathione(GSH)levels in Lim treated oocytes were significantly decreased(P<0.05)and increased(P<0.05)compared with control group.m RNA expression levels of antioxidant and apoptosis-related genes in bovine oocytes were detected by RT-q PCR.It was found that the m RNA expressions of SOD1,SOD3,SIRT2 and SIRT3 in the control group were significantly lower than those in the Lim group(P<0.05).Compared with the control group,the mitochondrial membrane potential(ΔΨm)of oocytes in Lim treatment group was significantly increased(P<0.05).Compared with the control group,the activity of cathepsin B(CB)in oocytes treated with Lim was significantly decreased(P<0.05).Compared with the control group,the m RNA expression of pro-apoptotic genes such as Caspase-3 and Bax were significantly decreased in Lim treatment group(P<0.05),while the m RNA expression of Survivin anti-apoptotic gene was significantly increased(P<0.05).In conclusion,Lim can effectively alleviate the damage of oxidative stress to oocytes,maintain mitochondrial function,inhibit apoptosis,improve the maturation quality of oocytes,and improve the development ability of oocytes.Experiment two: Effect of limonin on bovine early embryo developmentThe aim of this study was to investigate the effect of Lim on bovine early embryo development.Bovine oocytes treated with Lim were fertilized in vitro and developed into 8-cell embryos or blastocysts.By immunofluorescence staining of8-cell stage embryos,ROS level in control group was significantly higher than that in Lim group(P<0.05),GSH level was significantly lower than that in Lim group(P<0.05).The m RNA expressions of antioxidation-related genes such as SOD-1,SIRT-2and GPX-1 in the control group were significantly lower than those in the Lim group(P<0.05).Compared with the control group,the level of ΔΨm in Lim treatment group was significantly increased(P<0.05).Compared with the control group,the m RNA expression levels of zygotic genome activation-related genes OCT-4,COX-2 and SOX-2 in Lim treatment group were significantly increased(P<0.05).Compared with the control group,the number of cells in blastocyst in Lim group was significantly increased(P<0.05)and the apoptosis rate was significantly decreased(P<0.05),while the m RNA expression level of Bax pro-apoptotic gene in Lim group was significantly higher than that in the control group(P<0.05).The m RNA expressions of anti-apoptotic genes such as Bcl-2 and Survivin were significantly lower than those of Lim group(P<0.05).In conclusion,Lim can improve the development ability of early embryos and blastocyst quality by alleviating oxidative stress damage to early embryos,maintaining mitochondrial membrane potential,inhibiting cell apoptosis,up-regulating the expression of zygotic genome activation-related genes.In conclusion,this study suggests that Lim can effectively alleviate oxidative stress damage during in vitro maturation of bovine oocytes,improve in vitro maturation quality of bovine oocytes,and promote early embryo development. |
PDF Full Text Request