Comparison Of GDF-9and Smads Signal Pathway Between Bushen Treatment And Shugan Treatment In Vitro Culture Oocytes

Objective:With the development of society, the incidence of infertility isincreasing, the development of assisted reproductive technology is alsoimproving.Many new treatment such as IVF-ET,ICSI are more and moreapplied in clinical.But there are many obstacles during the therapy.Theresearch results of former clinical experiments have confirmed that Shugantreatment and Bushen Treatment could improve the number of eggs,thequality of eggs and the rate of fertilization and increase the rate of pregnancy.Bushen Tiaojing recipe and Xiaoyao Pill could also improve the protein levelof GDF-9in follicular fluid and the GDF-9mRNA expression in granule cells.Animal experiments confirmed that Bushen Tiaojing recipe and Xiaoyao Pillcould increase the number of oocyte,promote the release of oocytes andimprove the protein level of GDF-9and the expression of GDF-9mRNA.Prompting that Bushen treatment and Shugan treatment through amechanism of improve the GDF-9by means of the traditional Chinesemedicine.This study aimed to discuss the mechanisms and results between theBushen treatment and Shugan treatment during the in vitro culture of oocyte.Methods:28health female rats of6weeks old divided into7groups:Shugan Low dose group,Shugan Middle dose group,Shugan high dosegroup,Bushen low dose group,Bushen Middle dose group,Bushen high dosegroup,Control group.Xiaoyao pills was produced by Henan Wan Xipharmaceutical company.48pills dissolved in200ml distilled water.Thesuspension of Xiaoyao pills contains crude drugs0.18g/ml.Bushen TiaojingRecipe was purchased from Shijiazhuang Le Rentang drugstore, and wasmade into fine oral liquid by Department of Traditional ChineseMedicine.The suspension of Bushen Tiaojing recipe contains crude drugs1.62g/ml.The Control group were drenched with distilled water,Shugan low /middle/high groups were drenched with Xiaoyao pills, Bushen low/middle/high groups were drenched with Bushen Tiaojing Recipe twice aday,for1g/100g weight,2g/100g weight,3g/100g weight.Treatments continuedfor4days.The artery picks the blood in1h after the last dose.Standing2hsindoor,and centrifuge for3000rpm,30min,get supernatant for futuredetermination.Stored in20℃.432healthy female KunMing mice of12days old were used for3times.Every time144mice were used for experiment.They were randomlydivided into8groups:Control group,Normal group,Shugan low dosegroup,Shugan Middle dose group,Shugan High dose group,Bushen Low dosegroup,Bushen Middle dose group,Bushen High dose group.Separation of preantral follicles:Use the method of microdissection.Putthe mice to death with cervical dislocation.Get out two ovaries.Separation thefollicles under the stereo microscope.Choose the follicles of80-120μm.The in vitro culture of preantral follicles:Put2ml nutrient solution in35mm culture dish,37℃,5%CO2(V/V),100%humidity.After24hs thefollicles adherence to the dish.Add medicated serum to the solution.Classifythe follicles into8groups. Change1/2solution for every other day.Culturedfor6days.Separate the oocytes with enzyme.The first batch were put intoliquid nitrogen.The second and third batches were put into2%paraformaldehyde.The oocytes of the mice were got as above.Some were fixed in2%paraformaldehyde solution for immunohistochemistry,and the other werepreserved at-80℃.The protein content of GDF-9, BMPR II, ALK5, Smad2,Smad3and Smad4were detected by immunofluorescence andimmunohistochemistry. The mRNA expressions of them were analyzed byreal-time fluorescent quantitative PCR.Results:1In D2,the follicle adhere to the culture dish.In D4,the number of granulenumber increased.In D6,the granule number grow beyond the follicular theca.2Comparison of the survival follicle number in vitro culture In D2,the follicle number of Shugan high dose group and Bushen Highdose group was more than other groups,but the difference was not significant(P>0.05).In D4,the follicle number of Bushen high dose group was more thanother groups,and the difference was statistically significant(P<0.05).In D6,compare with control group,the follicle number of normal group was morethan control group,the difference was significant(P<0.05).The number offollicles in Bushen groups and Shugan groups was more than normal andcontrol groups,the Bushen high dose group was the highest one,the differencewas statistically significant(P<0.05).3Comparison of the expression of GDF-9,BMPR Ⅱ, ALK5, Smad2,Smad3,Smad4mRNA among groups in vitro culture of follicles.3.1The expression of GDF-9,BMPRⅡ,ALK5,Smad2,Smad3,Smad4mRNAat different points in timeIn Control and Normal groups,compare the expression of GDF-9,BMPRⅡ, ALK5, Smad2, Smad3, Smad4mRNA in D2,the expression in D4and D6was lower,the expression in D6was the lowest(P<0.05).Between each dosegroup of Shugan groups,the expression of GDF-9,BMPRⅡ, ALK5mRNA inD6was lower than that in D4and D2.Smad2, Smad3, Smad4mRNAexpression in D2,D4,D6has no significant difference between eachgroups.Between each dose group of Bushen groups,the expression ofGDF-9,BMPRⅡ, ALK5mRNA in D6was lowest(P<0.05).The expression ofSmad2, Smad3, Smad4mRNA has no significant difference between Busheneach groups(P>0.05).3.2The difference of the expression of GDF-9,BMPRⅡ, ALK5, Smad2,Smad3,Smad4mRNA between the groups.In the D2, D4, D6of the in vitro culture:The expression of GDF-9,BMPR Ⅱ, ALK5, Smad2, Smad3,Smad4mRNA in Normal group was higher than that in Control group,the differencewas not statistically significant(P>0.05).Compare with Control group and Normal group,the expression ofGDF-9,BMPRⅡ, ALK5, Smad2, Smad3,Smad4mRNA in Shugan groups was higher,the difference was statistically significant.The expression inShugan middle dose was the highest(P<0.05).Compare with Control groupand Normal group, the expression of GDF-9,BMPRⅡ, ALK5, Smad2,Smad3,Smad4mRNA in Bushen groups was higher, the difference wasstatistically significant.The expression in Bushen high dose were increasedsignificantly (P<0.05).Compare the expression of GDF-9,Smad2, Smad3,Smad4mRNAbetween Shugan groups and Bushen groups,the expression in Bushen highdose was increased statistically significant(P<0.05). The following wasShugan middle dose,Bushen middle dose,Shugan high dose.The expression ofBMPR Ⅱ mRNAin Shugan middle dose was increasedsignificantly(P<0.05).The expression of ALK5mRNA in Bushen high dosewas increased significantly (P<0.05).4Comparison of the expression of GDF-9,BMPR Ⅱ, ALK5, Smad2,Smad3,Smad4protein among groups in vitro culture of follicles.4.1The expression of GDF-9,BMPRⅡ,ALK5,Smad2,Smad3,Smad4proteinat different points in timeCompare the expression of GDF-9,BMPRⅡ, ALK5, Smad2, Smad3,Smad4protein in D2in Control and Normal groups,the expression in D4andD6was lower than D2,the expression in D6was the lowest(P<0.05).Betweeneach dose group of Shugan groups, the expression of GDF-9,BMPRⅡ,ALK5,Smad4protein in D6was lower than that in D4and D2.Smad2,Smad3protein expression in D2,D4,D6has no significant difference betweeneach groups(P>0.05).Between each dose group of Bushen groups,theexpression of GDF-9,BMPRⅡ, ALK5protein in D6was lowest(P<0.05).Theexpression of Smad2, Smad3, Smad4protein has no significant differencebetween Bushen each groups(P>0.05).4.2The difference of the expression of GDF-9,BMPRⅡ, ALK5, Smad2,Smad3,Smad4protein between the groups.In the D2, D4, D6of the in vitro culture: The expression of GDF-9,BMPR Ⅱ, ALK5, Smad2, Smad3,Smad4protein in Normal group was higher than that in Control group,the differencewas statistically significant(P<0.05).Compare with Control group and Normal group,the expression ofGDF-9,BMPRⅡ, ALK5, Smad2, Smad3,Smad4protein in Shugan groupswas higher,the difference was statistically significant(P<0.05).The expressionin Shugan high dose was the highest(P<0.05).Compare with Control groupand Normal group, the expression of GDF-9,BMPRⅡ, ALK5, Smad2,Smad3,Smad4protein in Bushen groups was higher, the difference wasstatistically significant(P<0.05).The expression in Bushen high dose wereincreased significantly (P<0.05).Compare the expression of GDF-9,Smad2, Smad3,Smad4proteinbetween Shugan groups and Bushen groups,the expression in Bushen highdose was increased statistically significant(P<0.05). The following wasShugan high dose, Bushen middle dose,Shugan middle dose,Bushen low dose,Shugan low dose.The expression of BMPRⅡprotein in Shugan high dose wasincreased significantly(P<0.05).The expression of ALK5in Bushen high dosewas increased significantly (P<0.05).Conclusions:1Xiaoyao Pill and Bushen Tiaojing Recipe could increse the survivalfollicle number.As the time went on,the impact was more significant.2In the progress of in vitro culture of follicles, the expression of GDF-9,BMPRⅡ,ALK5mRNA and protein were on a declining curve.Promoting thatthe GDF-9and its receptor plays an important role in initial progress of thefollicle development.3The GDF-9,BMPRⅡ,ALK5mRNA and protein were on a decliningcurve during the progress of culture.But theXiaoyao Pill and Bushen TiaojingRecipe could increase the GDF-9,BMPRⅡ,ALK5mRNA and protein expressin each day of the culture.The mechanism of action was activate receptorALK6through BMPRⅡand other type2receptor, and then start the signalconduction of Smads pathway. 4The impact of GDF-9of Bushen Tiaojing Recipe in a dose-dependentmanner.5During the progress of culture,the BMPRⅡ mRNA and proteinexpression of Shugan middle dose was highest,the ALK5mRNA and proteinexpression was highest.The GDF-9mRNA and protein expression of BushenTiaojing recipe was highest. Both Xiaoyao pill and Bushen Tiaojing Recipecould increase oocyte number of the mice in vitro culture and expression ofGDF-9, but the mechanism between them is different.6Both Xiaoyao Pill and Bushen Tiaojing Recipe could increase thequality of oocytes,the impact was signigicant in the initial stage of folliculardevelopment.