Antioxidant Effect And Mechanism Of Genistein In Oocyte Vitrification

Objectives:Oocyte vitrification technology helps preserve female fertility and is a research hotspot in the field of assisted reproductive technology(ART).The fertilization ability and developmental potential of oocytes are generally reduced after vitrification,and the clinical application is hindered,but the precise molecular mechanism leading to the decline of oocyte quality after cryopreservation is still not clear.The study found that the main cause of this phenomenon may be due to oxidative damage of oocytes after vitrification,and efforts to solve oxidative damage have become a key link to improve the efficiency of vitrification.In recent years,it has been found that genistein are closely related to antioxidant activity,and its chemical structure can directly act as an antioxidant,and can regulate the expression of antioxidant enzyme genes and apoptosis-related genes to exert antioxidant effects.This study focused on the addition of genistein during vitrification,the survival of oocytes after cryopreservation,fertilization rate and developmental potential,reactive oxygen species(ROS)levels,mitochondrial membrane potential(MMP)levels,and superoxide The expression of dismutase(SOD1),glutathione peroxidase(GPx1)and caspase-3(Caspase-9)further analyzed the antioxidation and mechanism of genistein.Methods:Take 6-8 weeks old healthy Kunming female mice.The ovulation induction drugs were induced by routine intraperitoneal injection.The collected oocytes were randomly divided into four groups,fresh group and pure vitrification group(G0 Group),Genistein group(G10 group,G100 group,G200 group respectively represent 10μM,100μM,200μM concentration gradient),Solvent group(Solvent group).After different treatments according to the experimental design,each group of oocytes was cultured,incubated for 2hours after cryopreservation,and the survival rate of oocytes in each group was observed and recorded.The fertilization of oocytes in each group was recorded 1 day after in vitro fertilization(IVF),and the blastocyst formation of oocytes in each group was recorded 4.5days after IVF.After ROS and JC-1 staining,the concentration of ROS and MMP in each group of oocytes was compared according to the relative ratio of relative fluorescence ratio and red/green fluorescence intensity.The m RNA expression levels of SOD1,GPx1,Caspase-3 and Caspase-9 were detected by real-time PCR.Results1.Changes in survival rate of the four groups of oocytes.The survival rate of oocytes in group G0 was lower than that in Fresh group,and the difference was statistically significant.Compared with G100 group,the survival rate of oocytes in G0 group,G10 group,G200 group and Solvent group was significantly lower,and the difference was statistically significant.2.Changes in fertilization rate and developmental potential of oocytes after IVF.Compared with the Fresh group,the fertilization rate,2-cell rate and blastocyst formation rate in G0 group,G10 group,G200 group and Solvent group were significantly lower,and the difference was statistically significant.In the vitrification groups,the G100 group had the best in vitro development potential,and the difference was statistically significant.3.Changes in ROS of the four groups of oocytes.The results of laser confocal microscopy and relative fluorescence ratio showed that the ROS level of oocytes in G0 group was significantly higher than that in Fresh group and G100 group.Compared with Fresh group,ROS levels in oocytes of G0 group,G100 group and Solvent group were increased,and the most obvious in G0 group,the difference was statistically significant.4.Changes in MMP of four groups of oocytes.The results of laser confocal microscopy and the relative intensity of red/green fluorescence showed that the MMP level in Fresh group oocytes was significantly higher than that of G0,G100 and Solvent group oocytes,and the MMP levels of G0 group and Solvent group were significantly lower.The difference was statistically significant.5.Expression of SOD1,GPx1,Caspase-3 and Caspase-9 in four groups of oocytes.Compared with the Fresh group,the expression levels of SOD1 and GPx1 m RNA in G0 group were significantly lower,while the expression levels of SOD1 and GPx1 m RNA in G100 group were higher than those in G0 group.The difference was statistically significant.The expression levels of Caspase-3 and Caspase-9 m RNA in G0 group were significantly higher than those in Fresh group,while the expression levels of Caspase-3and Caspase-9 m RNA in G100 group were lower than those in G0 group.The difference was statistically significant.Conclusion1.Genistein increases the survival rate of oocytes after vitrification and resuscita-tion,and reduces the effects of cold stress and oxidative damage on oocytes during vitrification.2.Genistein can improve the developmental problems of low fertilization rate and poor cleavage activity of oocytes after vitrification and resuscitation by reducing oxidative damage.3.The hydrogen atom attached to the hydroxyl group on the benzene ring in the chemical structure of genistein can directly act as an antioxidant,and genistein can also exert antioxidant effects by binding to estrogen receptor(ERs),α and β subunits,indicating that genistein can reduce ROS levels in oocytes.4.Genistein reduce the damage of MMP by reducing the production of ROS,and protect the mitochondrial function,thus providing guarantee for the quality and metabolism of oocytes.5.Genistein can up-regulate the expression of SOD1 and GPx1,remove excess superoxide anion and promote the reduction of peroxide,and enhance the antioxidant capacity of oocytes during vitrification;by down-regulating Caspase-3 and Caspase-9 The expression inhibits caspase activation,thereby reducing oocyte apoptosis.