Comparative Study Of The Relationship About Bushen Treatment And Shugan Treatment On The Quality Of Mouse Oocytes By Regulating Oocyte-secreted Factors And Smads Signal Pathway

Infertility is a serious impact on human reproductive health, theincidence of about7%-10%in our country. As the rapid development ofassisted reproductive technology(ART), in vitro fertilization-enbryo transfer(IVF-ET) technology has more than90%chance of success in raising follicles,it can improve embryo quality and incrase the numbers of high qualityembryos, but the success rate still remains low with relatively high rate ofmultiple pregnancy and abortion, how to improve the quality and quantity ofoocytes in IVF-ET process has become the focus. In recent years, researcherson Traditional Chinese Medicine (TCM) used to induce ovulation, improvethe oocytes quality and pregnancy rates, and reduce the side effects of westernmedicine has made great progress. Our research team studied the effects ofBushen treatment and Shugan treatment on the reprodutive function ofanovulatory rats and conclued that they can promote the development ofuterine, the maturation of follicular, and tmediate the endometrial environmentfor yunkun implantation by improving the adenohypophysis ovarianmorphology and mediating neurotransmitter and reproductive hormones levels.We also studied the effects of Bushen Tiaojing Recipe and Xiaoyao Pill onoutcome of IVF-ET and its effects on oocye-secreted factors (OSFs) andconclued that they can promote the oocyte numbers, high quality embryo rateand clinical pregnancy rate.The quantity and quality of ovum directly affect the outcome of IVF-ET.In recent years, the study of reproductive physiology and endocrinology found:other than the rugulation of hormones secreted from hypothalamus andpitutitary, oocyte secretion factors (OSFs) can regulate follicle growth and recession, and affect the choice of dominant follicles and the formation ofatresia follicles. In the process, GDF-9and BMP-6(from transforming growthfactor-β) play an important role, they can start signal transduction by typingwith Ⅱ serine/threonine kinase receptors and Ⅰ receptors. Smads is the signaltransduction molecules belong to TGF-β superfamily, and play an importantrole in signal transduction process. GDF-9and BMP-6and the Smads signalpathways play an important role in ovarian follicles development and thespread of eggs and the oocyte maturation. Because of the limitation of theovum collection in clinical, in this study we observed the relationship ofBushen treatment and Shugan treatment on the quality of mice oocytes and theSmads signal pathways through mice OSFs and Smads signaling pathways inorder to discuss the mechanism and supply the experimental basis fortraditional chinese medicine used in assistant therapy for IVF-ET.Part one Effects of Bushen treatment and Shugan treatment on thequality of mouse oocytes and pregnancy outcomesObjective: To discuss Bushen treatment and Shugan treatment on theqiality of mouse oocytes and pregnancy outcomes by observing effects ofBushen Tiaojing Recipe and Xiaoyao Pill on the oocyte numbers, the highquality follicle numbers,the pregnancy rate, the litter size, the embryonicnumbers and the related hormones in mice.Methods: We choose120male mice of8-9weeks old who hasreproduction ability and210healthy female KunMing mice of6-7weeks old,the female mice were randomly divided into6groups: Bushen high dosegroup, Bushen low dose group, Shugan high dose group, Shugan low dosegroup, COH group and Control group (N=35). Bushen groups were drenchedwith Bushen Tiaojing fine oral liquid (high dose5.4g/ml, low dose2.7g/ml),Shugan groups were drenched with Xiaoyao suspension (high dose0.6g/ml,low dose0.3g/ml), the control group and COH group were drenchedwith distilled water once a day, we began to dose at8:00everyday,1ml/100gtreatments continued for10days. The treatment groups and the COH groupwere dealed with intraperitoneal injection of PMSG10IU/only on the eleventh day, after48hours intraperitoneal injection of HCG10IU/only, thecontrol group were observed the oestrous cycle from daily vaginal smear. Atthe same of HCG injection,20mice from each group (the control groupremaining a large number of keratin epithelial cells from vaginal smear) weremated with male mice according to1:1ratio, and were examinated femalevaginal in the next morning7:00-8:00, we put the day found Yin chuan as thefirst gestation day, and observe the numbers of pregnancy and uterus embryosafter18-20days.15mice in control group (the next day of remaining a largenumber of keratin epithelial cells from vaginal smear) and the rest mice (afterHCG injection for16h) were sacrificed to count the number of oocytes andthe high-quality follices after determining the levels of FSH, LH and E2inserum by taking blood from eye.Results:(1)Comparison of the oocyte numbers and the high-qualityfollices: Compared with the control group, the numbers of follicle of the COHgroup and the treatment groups increased (P＜0.05), compared with the COHgroup, the treatment groups were no statistical significance (P＞0.05), thenumbers of follicle among the treatment groups were no statisticalsignificance (P＞0.05). Compared with the control group and the COH group,the high-quality follicle numbers of the treatment groups were increasedsignificantly (P＜0.05), the high-quality follicle numbers of COH group werediscreased compared with the control group; comparison between thetreatment groups: the high-quality follicle numbers of the high dose groupwere higher than the low dose (P＜0.05), the numbers of high-quality folliclesin high dose groups were no statistical significance(P＞0.05), the numbers ofhigh-quality follicles in low dose groups were no statistical significance (P＞0.05).(2)Comparison of the pregnancy rate and uterus embryonic numbers ineach group: the pregnancy rate of the COH group was lower than the controlgroup (P＜0.05); compared with the control group and COH group, thepregnancy rate of the high dose groups increased significantly (P＜0.05), thepregnancy rate of the low dose groups were no statistical significancecompared with the control group (P＞0.05); the pregnancy rate of the high dose groups were higher than the low dose groups (P＜0.05), the pregnancyrate between the Bushen high group and Shugan high group (between theBushen low group and the Shugan low group) has no statistical significance(P＞0.05). the embryonic numbers among groups had no statisticalsignificance (P＞0.05).(3)Comparison of FSH, LH and E2in serum in eachgroup: Compared with the control group, the levels of FSH, LH and E2of theCOH group and the treatment group were increased (P＜0.05), compared withthe COH group, the treatment groups were increased (P＜0.05), the levels ofFSH, LH and E2of high dose groups were higher than those of low dosegroups, comparison among high dose groups had no statistical significance e(P＞0.05), comparison among low dose groups had no statistical significancetoo (P＞0.05).Conclusions: Bushen Tiaojing Recipe and Xiaoyao Pill can not reduceoocyte numbers, but increase the high-quality oocyte numbers, improve thepregnancy rate and the total number of embryos in mice treated withgonadotropins.Part two Comparison of the effects of Bushen treatment andShugan treatment on GDF-9and Smads pathways of the mouse oocytesObjective: To investigate the effects of Bushen treatment and Shugantreatment on GDF-9, BMPRII, ALK-5, Smad2and Smad3of mouse oocytesand to explore the mechanism of Bushen treatment and Shugan treatment onthe quality of oocytes.Methods:600healthy female KunMing mice of6-7weeks old wererandomly divided into6groups: Bushen high dose group, Bushen low dosegroup, Shugan high dose group, Shugan low dose group, COH group andcontrol group (N=100). Bushen groups were drenched with Bushen Tiaojingfine oral liquid (high dose5.4g/ml, low dose2.7g/ml), Shugan groups weredrenched with Xiaoyao suspension (high dose0.6g/ml, low dose0.3g/ml), thecontrol group and COH group were drenched with distilled water once a day,We began to dose at8:00everyday,1ml/100g treatments continued for10days. The treatment groups and the COH group were dealed with intraperitoneal injection of PMSG10IU/only on the eleventh day, after48hours intraperitoneal injection of HCG10IU/only, the control group wereobserved the oestrous cycle from daily vaginal smear.16hours afterintraperitoneal Injection of HCG, the mice of the COH group and thetreatment groups were killed, the mice of the control group were killed on theday after a large number of keratin epithelial cells from vaginal smear, thenwe pierced the ampulla of fallopian tube with1ml syringe needle by stereomicroscope, collected cumulus oocyte complexes (COCs), digested cumulategranulosa cells with the hyaluronidase, and washed them by PBS again andagain. Finally we obtained MII oocytes for experiment, The protein andmRNA expression of GDF-9, BMPR II, ALK5, Smad2and Smad3weredetected by immunohistochemistry and real-time reverse transcription PCR.The protein content of GDF-9and BMPR II in oocytes were analyzed byWestern blot.Results:(1)Compared with the control group, the protein content andmRNA expression of GDF-9in the COH group were discreased (P＜0.05),the treatment groups were increased (P＜0.05); the protein content andmRNA expression of GDF-9in high dose group higher than in low dosegroups (P＜0.05), while there were no statistical significance between Bushenhigh dose group and Shugan high dose group (P＞0.05), the same as thatbetween Bushen low dose group and Shugan low dose group (P＞0.05).(2)Compared with the control group, the protein content and mRNAexpression of BMPR II in other groups had no statistical significance (P＞0.05), the protein content and mRNA expression of BMPR II in Shugan highgroup increased compared with other five groups (P＜0.05).(3)The proteincontent and mRNA expression of ALK-5, Smad2and Smad3were nostatistical significance among the groups (P＞0.05).Conclusions: Bushen Tiaojing Recipe and Xiaoyao Pill both canimprove the protein and mRNA expression of GDF-9in mice oocytes, andthus improve folllicular development and the quality of oocytes, but themechanism has nothing to do with the GDF-9receptor Ⅰa nd Smadssignal pathway.Part three Comparison of the effects of Bushen treatment andShugan treatment on BMP-6and Smads pathways of the mouse oocytesObjective: To investigate the effects of Bushen treatment and Shugantreatment on BMP-6, ALK-2, ALK-6, Smad1, Smad5, Smad8and Smad4ofmouse oocytes and to explore the mechanism of Bushen treatment and Shugantreatment on the quality of oocytes.Methods: Group and materials are the same as that in part two.Immunohistochemistry and Western blot were used to detect the proteincontent of BMP-6, ALK-2, ALK-6, Smad1, Smad5, Smad8and Smad4, themRNA expression of these targets were analyzed by Real-time reversetranscription PCR.Results:(1)BMP-6: compared with the control group, the protein andmRNA expression in the COH group had no statistical significance (P＞0.05),the treatment groups increased (P＜0.05);compared with the treatmentgroups, the expression of BMP-6in the COH group decreased (P＜0.05). Theprotein and mRNA expression in Bushen high dose group were higher thanthe Shugan group and the Bushen low group (P＜0.05), the protein andmRNA expression in Shugan high group increased compared with the Bushenlow group and the Shugan low group (P＜0.05), while there were nostatistical significance between the Shugan low group and the Bushen lowgroup (P＞0.05).(2)ALK-2: there were no statistical significance between thecontrol group and the COH group (P＞0.05), compared with the control groupand the COH group, the expression of ALK-2in the treatment groupsincreased (P＜0.05). The protein and mRNA expression in high dose groupwere high than the low dose group (P＜0.05), the protein and gene expressionin Shugan high group discreased compared with the Bushen high group (P＜0.05), there were no statistical significance between the Shugan low group andthe Bushen low group (P＞0.05).(3)ALK-6: compared with the control group,the protein and mRNA expression in the COH group had no statisticalsignificance (P＞0.05); compared with the treatment groups, the protein and mRNA expression of ALK-6in the control group and the COH groupdiscreased (P＜0.05); Bushen group expressed higher than Shugan group (P＜0.05), the protein and mRNA expression in the Bushen high dose groupwere higher than those in the Bushen low dose group (P＜0.05), the Shuganhigh dose group expressed higher than the Shugan low dose group (P＜0.05).(4)Smad1: there were no difference between the control group and the COHgroup (P＞0.05); the protein and mRNA expression of Smad1in treatmentgroups were higher than those in the control group and the COH group (P＜0.05); compared with the Bushen high dose group, the other treatment groupdiscreased (P＜0.05), the protein and mRNA expression in Bushen low dosegroup were higher than those in Shugan group (P＜0.05), Shugan high dosegroup expressed higher than the Shugan low dose group (P＜0.05).(5)Smad5:compared with the norma group, the COH group had no statisticalsignificance (P＞0.05), the protein and mRNA expression in the treatmentgroups increased, and higher than the COH group (P＜0.05); the protein andmRNA expression of Smad5in high dose groups were higher than those inlow dose groups (P＜0.05), and Bushen high dose group increased comparedwith Shugan high dose group (P＜0.05), Shugan low dose group discreasedcompared with Bushen low dose group (P＜0.05).(6)Smad8: there were nostatistical significance between the control group and the COH group (P＞0.05); the protein and mRNA expression of Smad8in treatment groups werehigher than those in the control group and the COH group (P＜0.05);compared with the high dose groups, the protein and mRNA expression in lowdose groups discreased (P＜0.05), while there were no statistical significancebetween the Shugan high dose group and the Bushen high dose group (P＞0.05), the protein and mRNA expression in Bushen low dose group increasedcompared with the Shugan low dose group (P＜0.05).(8)Smad4: comparedwith the control group, the gene and protein expression in the COH group hadno statistical significance (P＞0.05), the expression of Smad4in the treatmentgroups increased (P＜0.05). The protein and mRNA expression in high dosegroups were higher than the low dose groups (P＜0.05), the protein and mRNA expression in Bushen high group increased compared with the Shuganhigh dose group (P＜0.05), while there were no statistical significancebetween the Shugan low dose group and the Bushen low dose group (P＞0.05).Conclusions: Xiaoyao Pill can improve the expression of BMP-6inmouse oocytes, raise a second type of receptor BMPRⅡ, activate receptorsALK-2/6, and start the signal transduction of Smads pathway; BushenTiaojing Recipe may activate receptors ALK-2/6trough others type2receptors, and start the signal transduction of Smads pathway; the mechanismbetween them is different.