Study On Bushen Tiaochong Recipe To Improve Mouse Oocyte Quality By Inhibiting ROS

Objective: Taking ovarian oxidative stress as a model,the preventation effect of traditional Chinese medicine Bushen Tiaochong Recipe on mouse oocyte quality and the anti-oxidation pathway of mouse oocyte were studied.Method: A total of seventy-five SPF grade 4 to 5 weeks old ICR female mouse were randomly divided into control group,model group and Chinese medicine group.25 mice are in each group.The control group was continuously intragastrically administered with 20 mg/kg/d saline for 4 weeks,and daily intraperitoneal injection of sterile saline was started for 1 week in the last week of intragastric administration.The model group was continuously intragastrically administered with 20 mg/kg/d saline for 4 weeks,and daily intraperitoneal injection of 12.5 mg/kg 3-nitropropionic acid(3-NPA)was started for 1 week in the last week of intragastric administration.The Chinese medicine group was continuously intragastrically administered with 20mg/kg/d of Bushen Tiaochong Recipe for 4 weeks,12.5 mg/kg 3-NPA was intraperitoneally injected daily for 1 week in the last week of intragastric administration.On the 26 th day of continuous intragastric administration,0.2 ml pregnant horse serum(PMSG)was intraperitoneally injected,and 48 hours later,0.2 ml human chorionic gonadotropin(hCG)was intraperitoneally injected.Oocytes and ovaries were collected 14 to 16 hours after the injection of hCG.Weigh the body weight and ovary weight of the mice,and calculate the weight gain and ovarian index of the mice.Observe whether the obtained oocytes extruded the first polar body,and calculate the number of obtained oocytes and the first polar body extrusion rates.The activity of SOD,GSH-Px and CAT in ovarian tissue was detected.The level of ROS in MII oocytes was detected by DCFH-DA.The distribution of mitochondria in MII oocytes was observed by mitochondrial fluorescent probe(Mito-Tracker Green).The MII oocyte cortical granules were stained with FITC-PNA and their distribution was observed.The MII oocyte RNA was extracted and the expression levels of Sirt1,FOXO3 a and Mn-SOD were detected by RT-PCR.Results: 1.Mouse weight gain and ovarian index: There was no significant difference in weight gain among the three groups(P>0.05).In comparation with the control group and the Chinese medicine group,the ovarian index of the model group decreased significantly(P<0.05).In addition,there was no significant difference in the ovarian index between the Chinese medicine group and the control group(P>0.05).2.The number of mouse oocytes and extrude rate of the first polar body: In comparation with the control group and the Chinese medicine group,the number of oocytes obtained in the model group was significantly decreased(P<0.05),but the number of eggs obtained between the Chinese medicine group and the control group was not significant difference(P>0.05).Compared with the control group,the difference in the first polar body extrusion rates in the model group and the Chinese medicine group was statistically significant(P<0.05).In addition,the first polar body extrusion rates in the model group was significantly lower than that in the Chinese medicine group(P<0.05).3.ROS levels in MII oocytes: In comparation with the control group and the Chinese medicine group,the ROS levels in the oocytes of the model group were significantly increased(P<0.05).However,there was no significant difference in ROS levels between the control group and the Chinese medicine group(P>0.05).4.The activity of T-SOD,GSH-Px and CAT in mouse ovarian tissue: In comparation with the control group and the Chinese medicine group,the levels of T-SOD,GSH-Px and CAT in the ovarian tissue of the model group were significantly increased(P<0.05).Compared with the control group,the T-SOD level of the ovarian tissue of the Chinese medicine group was also significantly increased(P<0.05),and the levels of GSH-Px and CAT were not significantly increased(P>0.05).5.The distribution of mitochondria in MII oocytes: In comparation with the control group and the Chinese medicine group,the mitochondria of the oocytes in the model group accounted for a large proportion,and the difference was statistically significant(P<0.05).There was no significant difference in the distribution of mitochondria between the control group and the Chinese medicine group(P>0.05).6.The distribution of cortical granules in MII oocytes: In comparation with the control group and the Chinese medicine group,the distribution of cortical granules in the model group was more than that in the III level,and the difference was statistically significant(P<0.05).There was no significant difference in the distribution of cortical particles between the control group and the Chinese medicine group(P>0.05).7.The expression levels of Sirt1,FOXO3 a and Mn-SOD in MII oocytes: The expression of Sirt1 mRNA,FOXO3 a mRNA and Mn-SOD mRNA in the model group and the Chinese medicine group were significantly higher than those in the control group(P<0.05).Compared with the model group,the expression of Sirt1 mRNA and FOXO3 a mRNA in the oocytes of the Chinese medicine group were significantly decreased(P<0.05),but there was no significant difference in the expression of Mn-SOD mRNA(P>0.05).Conclusion: The model of ovarian oxidative stress induced by 3-NPA can lead to a decrease in ovarian reserve function,which is manifested by a decrease in the number of oocytes obtained after ovulation induction and an abnormality in oocyte nuclear maturation and cytoplasmic maturation.Pre-supplement of Bushen Tiaochong Recipe can protect the ovaries from oxidative damage and improve oocyte quality.In addition,Sirt1 in mouse oocytes may stimulate Mn-SOD expression by deacetylation of FOXO3 a,thereby reducing ROS levels.