The Effects Of Psychological Stress On Oxidative Stress Of Mice Oocytes And Early Embryonic Development Based On The Theory Of "Infertility Due To Jealousy"

Objective（1）By observing the body weight,open field test and sucrose preference test of female mice stimulated by different degrees of psychological stress,we can confirm whether the model of psychological stress was successfully established.Oocytes were collected during ovulation induction and the levels of ROS,SOD₂、CAT and GSH-Px m RNA in oocytes were detected to study the effects of different degrees of psychological stress on the oxidative stress level of oocytes in mice.（2）Mice were fertilized in vivo and fertilized eggs were collected for in vitro culture.In order to study the effects of different degrees of psychological stress on early embryonic development in mice,and to provide new ideas for the clinical treatment of female reproductive function decline from the perspective of liver depression and psychological stress,2-cell rate,8-cell rate,blastocyst formation rate,blastocyst hatching rate,blastocyst classification and the degree of blastocyst apoptosis were measured.MethodsExperiment 1:Study on oxidative stress level of mice oocytes induced by psychological stress80 ICR mice of 4～5 weeks old were selected.They were randomly divided into four groups:normal group,psychological stress strong stimulation group,psychological stress middle stimulation group and psychological stress weak stimulation group.The normal group did not give any intervention,the experimental group according to the preparation of the psychological stress model,given different degrees of unpredictable stimulation,for 6 weeks.The body weight of mice in each group was recorded before and after the stimulation;the changes of static time,exercise time,total distance of exercise,central activity time and peripheral activity time of mice in open field within 5 minutes after the stimulation;and the sucrose preference index of mice in each group after the stimulation to confirm the successful preparation of the model of psychological stress.After successful modeling,oocytes were obtained by ovulation induction in mice.The ROS concentration in mice oocytes was determined by chemiluminescence method,and the m RNA levels of SOD₂、CAT and GSH-Px in oocytes were determined by RT-PCR,in order to study the effects of different degrees of psychological stress on the oxidative stress level of oocytes in mice.Experiment 2:Study on early embryonic development of mice induced by psychological stressAccording to experiment 1,after the successful establishment of the model,the mice were fertilized in vivo,and the fertilized eggs were collected and processed after the examination of the vaginal plug in the next morning.In order to study the effects of different degrees of psychological stress on early embryonic development of mice,the number of fertilized eggs,the number of 2-cell embryos,the number of 8-cell embryos,the number of blastocysts and morphological scores of the mice in each group were observed under the microscope,and the rate of 2-cell,8-cell,the rate of blastocysts formation,the rate of blastocysts hatching and the grading of blastocysts were recorded.TUNEL was used to detect the degree of apoptosis of mice blastocysts.ResultsAll experimental data were analyzed by SPSS24.0 statistical software.The quantitative data were expressed as mean±standard deviation（^<sub>x±s）.One-way ANOVA was used for the comparison between groups with homogeneous variance,and LSD test was used for the pairwise comparison between groups.Welch’s ANOVA was used for the comparison between groups when the variance was uneven,and Games-Howell test was used for the pairwise comparison between groups.Kruskal-wallis H test was used for comparison between hierarchical data groups and pairwise comparison.P<0.05 was statistically significant.Experiment 11.Comparison of body weight of mice before and after stimulationThere was no significant difference in body weight between the four groups before stimulation（P>0.05）;After 6 weeks of stimulation,the body weight of mice in the psychological stress strong stimulation group,the middle stimulation group and the weak stimulation group were lower than that in the normal group,and the difference was statistically significant（P<0.05）;Further comparison between the two groups showed that the body weight of mice in the middle stimulation group and the weak stimulation group was higher than that in the strong stimulation group（P<0.05）.2.Comparison of open field test results of mice in each group after 6 weeks of stimulationAfter 6 weeks of stimulation,the resting time and the peripheral activity time of mice in the psychological stress strong stimulation group,the middle stimulation group and the weak stimulation group were higher than those in the normal group,and the difference was statistically significant（P<0.05）;Further comparison between the two groups showed that the resting time of mice in the middle stimulation group and the weak stimulation group was lower than that in the strong stimulation group,and the difference was statistically significant（P<0.05）.After 6 weeks of stimulation,the exercise time,the central activity time and the total distance of movement in the psychological stress strong stimulation group,the middle stimulation group and the weak stimulation group were lower than those in the normal group,the difference was statistically significant（P<0.05）;The further comparison between the two groups showed that the exercise time and the total distance of movement in middle and weak stimulation group were higher than those in strong stimulation group（P<0.05）;The central activity time in the middle stimulation group was higher than that in the strong stimulation group（P<0.05）.3.Comparison of sucrose preference index of mice in each group after 6 weeks of stimulationAfter 6 weeks of stimulation,the sucrose preference index of mice in the psychological stress strong stimulation group,the middle stimulation group and the weak stimulation group were lower than that of mice in the normal group,and the difference was statistically significant（P<0.05）.4.Comparison of ROS concentration in oocytes of mice in each groupThe concentration of ROS in oocytes of mice in the psychological stress strong stimulation group,the middle stimulation group and the weak stimulation group were all higher than that in the normal group,and the difference was statistically significant（P<0.05）;Further comparison between the two groups showed that ROS concentration in mice oocytes increased with the increase of stimulation intensity,and the differences were statistically significant（P<0.05）.5.Comparison of expression levels of SOD₂、CAT and GSH-Px in mice oocytes of each groupThe SOD₂,CAT and GSH-Px expression level of mice oocytes in the psychological stress strong stimulation group,the middle stimulation group and the weak stimulation group were all lower than those in the normal group,and the difference was statistically significant（P<0.05）;Further comparison between the two groups showed that the SOD₂,CAT and GSH-Px expression level in mice oocytes decreased with the increase of stimulation intensity,and the differences were statistically significant（P<0.05）.Experiment 21.Comparison of early embryonic development of mice in each groupThere were significant differences between the 2-cell rate,8-cell rate and the blastocyst formation rate of the four groups（P<0.05）.Further comparison between the two groups showed that the 2-cell rate of the mice in the psychological stress strong stimulation group was lower than that of the mice in the normal group,and the difference was statistically significant（P<0.05）by analysis of variance;The 8-cell rate and the blastocyst formation rate of mice in the psychological stress strong stimulation group and the middle stimulation group were lower than those of mice in the normal group,and the differences were statistically significant（P<0.05）;The 2-cell rate,8-cell rate and the blastocyst formation rate of the mice in the middle stimulation group and the weak stimulation group were higher than those of the mice in the strong stimulation group（P<0.05）.There was no significant difference in blastocyst hatching rate between the four groups（P>0.05）.2.Comparison of the results of blastocyst grading in each groupThere was significant difference in blastocyst grading between the four groups（P<0.05）.The blastocysts of normal group mice,psychological stress strong stimulation group mice and the middle stimulation group mice were mainly of grade 4,while those of weak stimulation group mice were mainly of grade 2 and grade 4.Further comparison between the two groups showed that there was significant difference in blastocyst classification between the strong stimulation group and the normal group（P<0.05）;There was significant difference between the blastocyst classification of the normal group and the middle stimulation group（P<0.05）.3.Comparison of apoptosis degree of mice blastocysts in each groupThe fluorescence intensity of blastula apoptosis in the psychological stress strong stimulation group,the middle stimulation group and the weak stimulation group were higher than that in the normal group,and the difference was statistically significant（P<0.05）;Further comparison between the two groups showed that the fluorescence intensity of blastula apoptosis increased with the increase of stimulation intensity,and the differences were statistically significant（P<0.05）.Conclusion1.Psychological stress led to the increase of ROS levels in mice oocytes,which triggered the oxidative stress reaction in oocytes,damaged the antioxidant system of oocytes,reduced the contents of SOD₂、CAT and GSH-Px antioxidant enzymes in oocytes,and damaged the function of oocytes.2.Psychological stress led to the decline of early embryonic development ability in mice,which reduced the 2-cell rate,8-cell rate and blastocyst formation rate in early embryos,affected the proportion of blastocysts at all levels,and then affected the blastocyst development process,and enhanced the degree of blastocyst apoptosis,thus resulting in the decline of embryo quality.3.Psychological stress causes oxidative stress damage to oocytes,reduces the potential of early embryo development,and then leads to infertility,which is in common with the theory of"infertility due to jealousy"in traditional Chinese medicine.This study provides new ideas and methods for the clinical treatment of female infertility from the perspective of liver stagnation and psychological stress,and provides a scientific basis for the promotion and application of the theory of"emotional and emotional pathogenesis"in traditional Chinese medicine under the modern medical mode.