| Objective: Ovulatory disorder is the main cause of female infertility.Our studying team confirmed that the Bushen treatment and Shugan treatment can regulate ovarian function,promote follicular development,induce ovulation in the previous research.This study on the relationship between Bushen treatment? Shugan treatment and ovulation pathway regulated by PR is intended to reveal the effect and mechanism of ovulation induced by Bushen treatment and Shugan treatment.Methods: 40 health female rats of 6 weeks old divided into 5 groups:Bushen Tiaojing Recipe?high dose group,Bushen Tiaojing Recipe ? high dose,Xiaoyao high dose group,Xiaoyao low dose group,Control group.Healthy female Kun Ming mice of 10 days old were used for experiment.They were randomly divided into 4 groups: Blank group,Normal group,Shugan group,Bushen group.Separation of preantral follicles: Use the method of microdissection.The culture of follicles in vitro: 37?,5%CO2(V/V),100% humidity.Adding medicated serum to the solution.Classify the follicles into 4 groups.Bushen group and Shugan group were changed medicated serum to Bushen Tiaojing III high dose and Xiaoyao Pill low dose serum respectively in D11 and cultured for 1d.Collecting follicles or excreted COCs in D12 after epidermal growth factor,hCG addition 0,8,12,16 hs.Cultured D12: Observing and measuring the diameter of the follicles and oocytes by to find the average value of both.Sixteen hours after the addition of hCG,the survival rate of follicles,the formation rate of sinus cavities,and the escape rate of oocytes were observed.Immunofluorescence staining was used to detect the protein expression of PR-A,PPAR?,ADAMTS-1,ADAM 8,HIF-1? and ET-2 in follicles and COCs,and the expression of mRNA was detected by real-time fluorescent quantitative PCR.Result:1 Morphological and structural changes during follicular growthCultured D1,follicles adhere to the bottom of the petri dish and original three-dimensional structure began to collapse;the diameter of D4 follicles increased significantly,the granular cells increase significantly and broke through the basement membrane;part of the follicles formed sinus cavities and the granulosa cells differentiate into cumulus granulosa cell layer and membrane granulosa cell layer,granular cell area appeares transparent area in the D8;D12 follicular diameter increases significantly,sinus cavity increases,the cumulus-oocyte complex(COCs)can be seen in follicles;D12 addtion hCG 16 h,the COCs discharged,the oocyte escapes and the first polar body be discharged.2 Comparison of follicle and oocyte diameter,survival rate of follicles,formation rate of sinus cavity and escape rate of oocytes in vitro in all groupsComparison of follicular diameter cultured in vitro after hCG be added16 hs.There is no significant difference in follicle diameter between Normal group and Blank group(P>0.05).Compared with Blank group and Normal group,follicle diameter increase in Bushen group and Shugan group(P<0.05),Bushen group was superior to Shugan group(P<0.05).Comparation of oocyte diameter,there is no statistical significance between the groups(P>0.05).The comparison of the survival rate of follicles: compared with the Blank group,the Normal group is not statistically significant(P>0.05);compared with the Blank group and the Normal group,Bushen group and Shugan group increase(P<0.05),Bushen group is higher than Shugan group(P<0.05).The rate of sinus formation and escape of oocytes were compared:compared with the Blank group,the Normal group has no statistical significance(P>0.05);compared with the Blank group and the Normal group,the Bushen groups and Shugan groups are higher(P<0.05),Bushen group is higher than Shugan group(P<0.05).3 Expression of PR-A,PPAR?,ADAMTS-1,ADAM 8,HIF-1? and ET-2proteinin in follicles or excreted COCs cultured in vitro in D12 after addition of hCG3.1 Expression of PR-A,PPAR?,ADAMTS-1,ADAM 8,HIF-1? and ET-2proteinin follicles or excreted COCs cultured in vitro in D12 at various time points in the same group after addition of hCGBlank group: Compared with 0h,the protein expression of PPAR?,ADAMTS-1,ADAM 8,HIF-1? and ET-2 at 8h has no statistical significance(P>0.05),and the expression of PR-A protein increase(P<0.05).Compared with 8h,there is no significant difference in the expression of PR-A,PPAR?,ADAMTS-1,ADAM 8,HIF-1?,ET-2at 12h(P>0.05).Compared with 12 h,the expression of PR-A,PPAR?,ADAMTS-1,ET-2 protein at 16 h has no statistical significance(P<0.05),and the expression of ADAM8,HIF-1?increase(P<0.05).Normal group: Compared with 0h,at 8h,the expression of PR-A,PPAR?,ADAMTS-1,ADAM 8,HIF-1? and ET-2 at 8h had no statistical significance(P<0.05).The expression of PPAR?,ADAMTS-1,HIF-1?,and ET-2 protein at 12 h had no statistical significance(P<0.05),and the expression of PR-A and ADAM8 protein increase(P<0.05).Compared with 12 h,The expression of PR-A,PPAR?,ADAMTS-1,ADAM8,HIF-1? and ET-2 at 16 h is not statistically significant(P<0.05).Bushen group: The expression of PR-A,PPAR?,ADAMTS-1,ADAM 8,HIF-1?,and ET-2 protein in the Bushen group gradually increase at 0h,8h,12 h,and 16 h.The expression of PR-A,ADAMTS-1 and HIF-1? at 8h increase(P<0.05)and the expression of PPAR?,ADAMTS-1,HIF-1? and ET-2 is not statistically significant(P>0.05).Compared with 0h,8h,,the increase in protein expression at 12 h and 16 h are statistically significant(P<0.05),and there is no significant difference between 12 h and 16h(P>0.05).Shugan group: The expression of PR-A,PPAR?,ADAMTS-1,ADAM 8,HIF-1? and ET-2 gradually increase at 0h,8h,12 h,and 16 h.Compared with0 h,the increase of protein expression at 8h was statistically significant(P<0.05).Compared with 8h,the increase of protein expression at 12 h and16h is not statistically significant(P>0.05),and there is no statistical significance between 12 h and 16h(P>0.05).3.2 Expression of PR-A,PPAR?,ADAMTS-1,ADAM 8,HIF-1? and ET-2proteins at the same time point after culture of D12 with hCG0h: There is no significant difference in the expression of PR-A,PPAR?,HIF-1? and ET-2 protein between Blank group,Normal group,Bushen group and Shugan group(P>0.05).8h: Compared with the Blank group,the expression of PR-A,PPAR?,ADAMTS-1,ADAM 8,HIF-1? and ET-2 protein in the Normal group is not statistically significant(P>0.05);compared with the Blank group and the Control group,the expression of PR-A,PPAR?,ADAMTS-1,ADAM 8,HIF-1? and ET-2 protein in the Bushen group and Shugan group significantly increase(P<0.05);the Shugan group was higher than the Bushen group.There was no significant difference in the expression of ADAMTS-1protein between the Bushen group ? Blank group and the Control group(P<0.05).12h: Compared with the Blank group,the expression of PR-A,PPAR?,ADAMTS-1,ADAM 8,HIF-1? and ET-2 protein in the Normal group is not statistically significant(P>0.05);compared with the Blank group and the Control group,the expressions of PR-A,PPAR?,ADAMTS-1,ADAM 8,HIF-1?,and ET-2 in the Bushen group and Shugan group are significantly higher(P<0.05),and the Bushen group is higher than the Shugan group.16h: Compared with the Blank group,the expression of PR-A,PPAR?,ADAMTS-1,ADAM 8,HIF-1? and ET-2 protein in the Normal group is not statistically significant(P>0.05);compared with the Blank group and the Control group,the expressions of PR-A,PPAR?,ADAMTS-1,ADAM 8,HIF-1? and ET-2 in Bushen group and Shugan group elevate(P<0.05);expression of PR-A,PPAR?,ADAMTS-1,ADAM 8,HIF-1? protein betweenthe Bushen group and Shugan group is not statistically significant(P> 0.05),ET-2 protein expression in Shugan group was higher than Bushen group(P<0.05).4 Expression of PR-A,PPAR?,ADAMTS-1,ADAM 8,HIF-1?,ET-2 mRNA in cultured follicles or excreted COCs4.1 The expression of PR-A,PPAR?,ADAMTS-1,ADAM 8,HIF-1? and ET-2 mRNA in the same group culturedBlank group: The expression of PR-A,PPAR?,ADAMTS-1,ADAM 8,HIF-1? and ET-2 mRNA expression gradually increase from 0h,8h,12 h to16h.Compared with 0h,PR-A,PPAR?,ADAMTS-1 expression increase,the increase is statistically significant in 8h(P<0.05).The increase of expression of ADAM 8,HIF-1? and ET-2 is not statistically significant(P>0.05).Compared with 0h,8h,12 h mRNA,expression increase in 16 h is statistically significant(P<0.05),and there is no significant difference between 12 h and16h(P>0.05).Normal group: The expression of PR-A,PPAR?,ADAMTS-1,ADAM 8,HIF-1?,and ET-2 mRNA gradually increase at 0h,8h,12 h,and 16 h.There is no statistical difference at 8h,compared with 0h(P>0.05).Compared with 0h and 8h,the increase of mRNA expression at 12 h and 16 h is statistically significant(P<0.05),and is not statistically significant between 12 h and 16h(P>0.05).Bushen group: The expression of PR-A,PPAR?,ADAMTS-1,ADAM 8,HIF-1? and ET-2 mRNA gradually increase at 0h,8h,12 h,and 16 h.8h the increase of PR-A,PPAR?,ADAMTS-1,ADAM 8,HIF-1?,ET-2 expression is statistically significant(P<0.05).Compared with 0h and 8h,the mRNA expression increase at 12 h and 16 h is statistically significant(P<0.05),but there is no statistically significant between 12 h and 16h(P<0.05).Shugan group: The expression of PR-A,PPAR?,ADAMTS-1,ADAM 8,HIF-1? and ET-2 mRNA gradually increase at 0h,8h,12 h,and 16 h.Compared with 0h,the protein expression at 8h increase significantly(P<0.05).Compared with 8h,there is no statistically significant increase in mRNAexpression at 12 h and 16h(P>0.05),there was no significant difference between 12 h and 16h(P>0.05).4.2 Expression of PR-A,PPAR?,ADAMTS-1,ADAM 8,HIF-1?,ET-2 mR-NA at different time points after hCG addition0h: Compared with the blank group,the expression of PR-A,PPAR?,ADAMTS-1,ADAM 8,HIF-1? and ET-2 mRNA in Normal group is not statistically significant(P>0.05);compared with the Blank group and the Control group,the mRNA expression of PR-A,PPAR?,ADAMTS-1,ADAM8,HIF-1? and ET-2 in the Bushen group and Shugan group increase,but the difference between the Bushen group and Shugan group is not statistically significant(P>0.05).8h: Compared with the Blank group,the expression of PR-A,PPAR?,ADAMTS-1,ADAM 8,HIF-1? and ET-2 mRNA in the Normal group is not statistically significant(P>0.05);compared with the Blank group and the Control group,the mRNA expression of PR-A,PPAR?,ADAMTS-1,ADAM8,HIF-1? and ET-2 in the Bushen group and Shugan group increase significantly(P<0.05).The Shugan group is higher than the Bushen group(P<0.05).12h: Compared with the Blank group,the expression of PR-A,PPAR?,ADAMTS-1,ADAM 8,HIF-1? and ET-2 mRNA in the Normal group is not statistically significant(P>0.05);compared with the Blank group and the Control group,the mRNA expression of PR-A,PPAR?,ADAMTS-1,ADAM8,HIF-1? and ET-2 in Bushen group and Shugan group are significantly higher(P<0.05);the Bushen group is higher than the Shugan group(P<0.05).16h: Compared with the Blank group,the expression of PR-A,PPAR?,ADAMTS-1,ADAM 8,HIF-1? and ET-2 mRNA in the Normal group is not statistically significant(P>0.05);compared with the Blank group and Control group,mRNA expression of PR-A,PPAR?,ADAMTS-1,ADAM 8,HIF-1?,and ET-2 in the Bushen group and the Shugan group increase(P<0.05).The Bushen group is higher than the Shugan group but there is no significant difference between the two groups(P>0.05).Conclusion:1.Bushen Tiaojing Recipe II,III and Xiaoyao Pill containing serum can promote the growth of mouse follicles in vitro and induce ovulation.2.Both Bushen treatment Shugan treatment can up-regulate the ex-pression of PR and its regulated follicle fragmentation related factors,such as PPAR?,ADAMTS-1,ADAM 8,HIF-1?,ET-2 protein and mRNA in follicles and COCs,thereby inducing ovulation.3.The time of ovulation induced by the Bushen treatment Shugan treatment is different.During ovulation is the Bushen treatment plays a promoting role,but the Shugan treatment is before ovulation,and the effect of on ovulation Bushen treatment is better than Shugan treatment. |
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