补肾法、疏肝法对小鼠卵母细胞质量的影响与调控OSFs及其Smads通路关系的比较研究
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摘要
不孕不育症是严重影响人类生殖健康的世界性问题，在我国的发病率约为7%-10%，随着人类辅助生殖技术（assisted reproductive technology，ART）的快速发展，体外受精-胚胎移植（In vitro fertilization-embryotransfer，IVF-ET）技术在卵泡的募集和卵母细胞的获取上已经有90%以上的成功机会，胚胎质量明显优化，获取优质胚胎的数量亦明显增多，但是其临床妊娠率却一直未能尽如人意，且多胎妊娠发生率和流产率较高，如何更好的提高IVF-ET过程卵细胞的数量及质量成为众多学者关注的焦点。近年来，中医药在提高卵母细胞质量、诱导排卵、提高妊娠率及降低西药的毒副作用等方面取得了长足的发展。本课题组前期研究了补肾法、疏肝法对无排卵模型大鼠生殖功能的影响，证实补肾法、疏肝法可以通过改善腺垂体、卵巢形态，调节下丘脑神经递质水平以及生殖内分泌激素水平，促进卵泡发育和成熟，并可促进子宫发育，调节子宫内膜环境以有利于孕卵着床；研究了补肾调经方、逍遥散对IVF-ET妊娠结局及OSFs的影响，证实加用补肾调经方、逍遥散可以增加获卵数，提高优质胚胎率及临床妊娠率。本课题拟在前期研究的基础上，进一步通过动物实验研究补肾法、疏肝法对卵母细胞发育的影响，为在临床上联合IVF-ET治疗不孕症奠定理论基础。
卵母细胞的数量及质量直接影响到IVF-ET的结局，近些年对生殖生理及生殖内分泌的研究发现，卵泡发育除了受下丘脑、垂体分泌的相关激素水平调控外，卵母细胞分泌因子（Oocyte secreted factors，OSFs）在调节卵泡的发育和衰退，影响优势卵泡的选择以及闭锁卵泡的形成方面发挥重要作用，其中，卵母细胞分泌的转化生长因子（transforming growthfactor-β，TGF-β）超家族成员GDF-9与BMP-6通过与Ⅱ型丝氨酸/苏氨酸激酶受体结合，与Ⅰ型受体共同作用启动信号转导[1]，而Smads是TGF-β超家族下游信号转导分子，在其信号转导过程中发挥重要的作用。GDF-9和BMP-6及其Smads信号途径对体内卵泡发生发育、卵丘扩散、卵母细胞的成熟至关重要[2-3]。由于在临床上收集人卵母细胞用于实验研究的局限性，本课题拟在前期研究的基础上，以小鼠卵母细胞OSFs及其Smads通路为切入点研究补肾法、疏肝法对小鼠卵母细胞质量的影响与调控OSFs及其Smads通路关系，探讨补肾法、疏肝法提高卵母细胞质量、改善妊娠结局的机制和作用环节，为中医药辅助IVF-ET治疗不孕症提供实验依据。
第一部分补肾法、疏肝法对小鼠卵母细胞质量及妊娠结局的影响
目的：观察补肾调经方、逍遥丸对小鼠卵母细胞数量、优质卵泡数、妊娠率、子宫胚胎数及相关激素水平的影响，以探讨补肾法、疏肝法对小鼠卵母细胞质量及妊娠结局的影响。
方法：选用6-7周龄健康雌性昆明小鼠210只，8-9周龄有生育能力雄性小鼠120只，将雌性小鼠按随机数字表法完全随机分为6组，即补肾高剂量组、补肾低剂量组、疏肝高剂量组、疏肝低剂量组、控制性超排卵组（COH组）和空白对照组，每组35只。补肾高、低剂量组分别给予补肾调经方口服液5.4g/ml、2.7g/ml，疏肝高、低剂量组分别给予逍遥丸混悬液0.6g/ml、0.3g/ml，COH组和空白对照组用蒸馏水灌胃。每日8:00各组小鼠均以1ml/100g体重灌胃，连续10天。治疗组及COH组于第11日同时腹腔注射PMSG10IU/只，48h后腹腔注射HCG10IU，空白对照组每日阴道涂片，观察动情周期。注射HCG同时，补肾高低剂量组、疏肝高低剂量组和COH组各取20只；空白对照组阴道涂片见到大量角化上皮细胞时取20只，按雌∶雄=1∶1比例分别合笼饲养，次日早晨7:00-8:00检查雌鼠阴道，发现阴栓者记为妊娠第1天（阴栓为小鼠阴道分泌物与精液凝结而成），于妊娠第18-20天解剖雌鼠，观察妊娠鼠数及子宫胚胎数。空白对照组剩余15只小鼠于阴道涂片见到大量角化上皮细胞的次日、其余各组剩余小鼠于注射HCG16h后，用摘除眼球法取血，留取血清后测定FSH、LH、E2的含量；然后脱颈椎处死小鼠，留取卵母细胞，计数卵泡数量及优质卵泡数。
结果：（1）各组小鼠获卵数及优质卵泡数比较：与空白对照组比较，COH组及各治疗组获卵数增加（P＜0.05），各治疗组与COH组比较均无统计学意义（P＞0.05），各治疗组之间获卵数比较无统计学意义（P＞0.05）；各治疗组优质卵泡数高于空白对照组及COH组（P＜0.05），COH组低于空白对照组（P＜0.05）；高剂量组优质卵泡数均优于低剂量组（P＜0.05），补肾高剂量组与疏肝高剂量组的优质卵泡数比较无统计学意义（P＞0.05），补肾低剂量组与疏肝低剂量组比较无统计学意义（P＞0.05）。（2）各组小鼠妊娠率及子宫胚胎数比较：COH组小鼠妊娠率较空白对照组降低（P＜0.05），补肾高剂量组和疏肝高剂量组妊娠率较空白对照组及COH组升高（P＜0.05），低剂量组与空白对照组比较无统计学意义（P＞0.05）；高剂量组小鼠妊娠率均优于低剂量组（P＜0.05），高剂量组间比较无统计学意义（P＞0.05），低剂量组间比较无统计学意义（P＞0.05）；各组之间子宫胚胎数比较均无统计学意义（P＞0.05）。（3）各组小鼠血清FSH、LH、E2值比较：与空白对照组比较，各治疗组及COH组血清FSH、LH、E2水平增高（P＜0.05）；与COH组比较，各治疗组血清FSH、LH、E2水平增高（P＜0.05），其中高剂量组的表达优于低剂量组（P＜0.05），补肾高剂量组与疏肝高剂量组之间无统计学意义（P＞0.05），补肾低剂量组与疏肝低剂量组之间无统计学意义（P＞0.05）。
结论：在用促性腺激素处理小鼠的同时加用补肾调经方和逍遥丸不会降低获卵数，并能增加优质卵母细胞数量，提高妊娠率及总胚胎数。
第二部分补肾法、疏肝法对小鼠卵母细胞GDF-9及Smads通路影响的比较
目的：通过研究补肾法、疏肝法对小鼠卵母细胞中卵母细胞分泌因子GDF-9、相关受体BMPRII、ALK-5及下游Smads通路中Smad2、Smad3的影响，探讨两法对卵母细胞质量影响的靶向性机制。
方法：选用6-7周龄健康雌性昆明小鼠600只，按随机数字表法完全随机化分为6组，即补肾高剂量组、补肾低剂量组、疏肝高剂量组、疏肝低剂量组、COH组和空白对照组，每组100只。补肾高、低剂量组分别给予补肾调经方口服液，疏肝高、低剂量组分别给予逍遥丸混悬液，COH组和空白对照组用蒸馏水灌胃。每日8:00各组小鼠均以1ml/100g体重灌胃，连续10天。补肾高剂量组、补肾低剂量组、疏肝高剂量组、疏肝低剂量组及COH组于第11日同时腹腔注射PMSG10IU/只，48h后腹腔注射HCG10IU/只；空白对照组每日阴道涂片，观察动情周期。注射HCG16h后，取补肾高低剂量组、疏肝高低剂量组和COH组小鼠，以及空白对照组阴道涂片见到大量角化上皮细胞的次日小鼠，脱颈椎处死，在体视镜下用1ml注射针划破输卵管壶腹部，收集卵丘卵母细胞复合体(COCs)，用透明质酸酶消化掉卵丘颗粒细胞后用PBS反复冲洗，获得MⅡ期卵母细胞，采用免疫组化法、实时荧光定量PCR分别检测卵母细胞中GDF-9、ALK-5、BMPR II、Smad2、Smad3蛋白及其mRNA表达；采用Western blot方法检测卵母细胞中GDF-9和BMPR II蛋白的表达。
结果：（1）与空白对照组比较，COH组GDF-9蛋白及mRNA的表达降低（P＜0.05），各治疗组的表达升高（P＜0.05）；高剂量组GDF-9蛋白及mRNA表达高于低剂量组（P＜0.05），补肾高剂量组与疏肝高剂量组比较无统计学意义（P＞0.05），补肾低剂量组与疏肝低剂量组比较无统计学意义（P＞0.05）。（2）与空白对照组比较，COH组、补肾高剂量组、补肾低剂量组及疏肝低剂量组BMPR II蛋白及mRNA表达无统计学意义（P＞0.05）；疏肝高剂量组BMPR II蛋白和mRNA的表达较其余各组均增强，有统计学意义（P＜0.05）。（3）ALK-5、Smad2、Smad3蛋白表达在各组间比较均无统计学意义（P＞0.05）。
结论：补肾调经方和逍遥丸均可以提高小鼠卵母细胞GDF-9蛋白及mRNA的表达，从而改善卵泡发育，提高卵母细胞质量，但其机制与GDF-9Ⅰ型受体及下游Smads通路无关。
第三部分补肾法、疏肝法对小鼠卵母细胞BMP-6及Smads通路影响的比较
目的：通过观察补肾法、疏肝法对小鼠卵母细胞中卵母细胞分泌因子BMP-6、相关受体ALK-2、ALK-6及下游Smads通路中Smad1、Smad5、Smad8和Smad4的影响，探讨两法对卵母细胞质量的影响的靶向性机制。
方法：动物分组及取材同第二部分。免疫组化和Western blot方法检测卵母细胞BMP-6、ALK-2、ALK-6、Smad1、Smad5、Smad8、Smad4蛋白的表达，实时荧光定量PCR法检测卵母细胞上述指标mRNA的表达。
结果：（1）BMP-6：与空白对照组比较，COH组卵母细胞BMP-6蛋白和mRNA的表达无统计学意义（P＞0.05），各治疗组表达升高，并高于COH组（P＜0.05）；疏肝高剂量组的表达高于补肾低剂量组和疏肝低剂量组（P＜0.05），低于补肾高剂量组（P＜0.05）；补肾低剂量组与疏肝低剂量组之间比较无统计学意义（P＞0.05）。（2）ALK-2：与空白对照组比较，COH组ALK-2蛋白及mRNA表达无统计学意义（P＞0.05）；各治疗组ALK-2的蛋白及mRNA表达均高于空白对照组和COH组（P＜0.05）；组间比较显示：各治疗组高剂量组ALK-2蛋白及mRNA表达均高于低剂量组（P＜0.05）；疏肝高剂量组表达较补肾高剂量组降低（P＜0.05），补肾低剂量组与疏肝低剂量组比较则无统计学意义（P＞0.05）。（3）ALK-6：与空白对照组比较，COH组ALK-6蛋白及mRNA表达无统计学意义（P＞0.05）；空白对照组和COH组与治疗组比较，ALK-6蛋白及mRNA表达降低（P＜0.05）；补肾组表达高于疏肝组（P＜0.05），补肾低剂量组的ALK-6蛋白及mRNA表达较补肾高剂量组降低（P＜0.05），疏肝高剂量组蛋白表达高于疏肝低剂量组（P＜0.05）。（4）Smad1：空白对照组与COH组比较无统计学意义（P＞0.05）；各治疗组Smad1蛋白及mRNA表达高于空白对照组和COH组（P＜0.05）；与补肾高剂量组比较，其余三个治疗组的表达降低（P＜0.05），补肾低剂量组较疏肝组蛋白及基因表达增强（P＜0.05），疏肝高剂量组Smad1蛋白及基因表达较疏肝低剂量组增强（P＜0.05）。（5）Smad5：与空白对照组比较，COH组表达无统计学意义（P＞0.05），各治疗组蛋白及mRNA表达增加，并高于COH组（P＜0.05）；高剂量组表达较低剂量组升高（P＜0.05），补肾高剂量组的表达高于疏肝高剂量组（P＜0.05），疏肝低剂量组较补肾低剂量组表达降低（P＜0.05）。（6）Smad8：空白对照组与COH组的Smad8蛋白及mRNA表达无统计学意义（P＞0.05）；各治疗组Smad8蛋白及mRNA表达较空白对照组和COH组升高（P＜0.05）；与高剂量组比较，各低剂量组的Smad8蛋白及mRNA表达均降低（P＜0.05），补肾高剂量组与疏肝高剂量组之间比较无统计学意义（P＞0.05），补肾低剂量表达较疏肝低剂量组增强（P＜0.05）。（7）Smad4：与空白对照组比较，COH组蛋白及mRNA表达无统计学意义（P＞0.05），各治疗组表达增高，有统计学意义（P＜0.05）；高剂量组Smad4蛋白及基因表达高于低剂量组（P＜0.05），补肾高剂量组表达高于疏肝高剂量组（P＜0.05），补肾低剂量组与疏肝低剂量组比较无统计学意义（P＞0.05）。
结论：逍遥丸可能通过提高小鼠卵母细胞BMP-6的表达，募集其Ⅱ型受体BMPRⅡ并激活ALK-2/6受体，启动下游Smad通路的信号传导；补肾调经方可能是通过募集其他Ⅱ型受体并激活ALK-2/6受体，启动下游Smad通路的信号传导；二者的作用机制有所差异。
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 outcomes
Objective: 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 oocytes
Objective: 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 oocytes
Objective: 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.
卵母细胞的数量及质量直接影响到IVF-ET的结局，近些年对生殖生理及生殖内分泌的研究发现，卵泡发育除了受下丘脑、垂体分泌的相关激素水平调控外，卵母细胞分泌因子（Oocyte secreted factors，OSFs）在调节卵泡的发育和衰退，影响优势卵泡的选择以及闭锁卵泡的形成方面发挥重要作用，其中，卵母细胞分泌的转化生长因子（transforming growthfactor-β，TGF-β）超家族成员GDF-9与BMP-6通过与Ⅱ型丝氨酸/苏氨酸激酶受体结合，与Ⅰ型受体共同作用启动信号转导[1]，而Smads是TGF-β超家族下游信号转导分子，在其信号转导过程中发挥重要的作用。GDF-9和BMP-6及其Smads信号途径对体内卵泡发生发育、卵丘扩散、卵母细胞的成熟至关重要[2-3]。由于在临床上收集人卵母细胞用于实验研究的局限性，本课题拟在前期研究的基础上，以小鼠卵母细胞OSFs及其Smads通路为切入点研究补肾法、疏肝法对小鼠卵母细胞质量的影响与调控OSFs及其Smads通路关系，探讨补肾法、疏肝法提高卵母细胞质量、改善妊娠结局的机制和作用环节，为中医药辅助IVF-ET治疗不孕症提供实验依据。
第一部分补肾法、疏肝法对小鼠卵母细胞质量及妊娠结局的影响
目的：观察补肾调经方、逍遥丸对小鼠卵母细胞数量、优质卵泡数、妊娠率、子宫胚胎数及相关激素水平的影响，以探讨补肾法、疏肝法对小鼠卵母细胞质量及妊娠结局的影响。
方法：选用6-7周龄健康雌性昆明小鼠210只，8-9周龄有生育能力雄性小鼠120只，将雌性小鼠按随机数字表法完全随机分为6组，即补肾高剂量组、补肾低剂量组、疏肝高剂量组、疏肝低剂量组、控制性超排卵组（COH组）和空白对照组，每组35只。补肾高、低剂量组分别给予补肾调经方口服液5.4g/ml、2.7g/ml，疏肝高、低剂量组分别给予逍遥丸混悬液0.6g/ml、0.3g/ml，COH组和空白对照组用蒸馏水灌胃。每日8:00各组小鼠均以1ml/100g体重灌胃，连续10天。治疗组及COH组于第11日同时腹腔注射PMSG10IU/只，48h后腹腔注射HCG10IU，空白对照组每日阴道涂片，观察动情周期。注射HCG同时，补肾高低剂量组、疏肝高低剂量组和COH组各取20只；空白对照组阴道涂片见到大量角化上皮细胞时取20只，按雌∶雄=1∶1比例分别合笼饲养，次日早晨7:00-8:00检查雌鼠阴道，发现阴栓者记为妊娠第1天（阴栓为小鼠阴道分泌物与精液凝结而成），于妊娠第18-20天解剖雌鼠，观察妊娠鼠数及子宫胚胎数。空白对照组剩余15只小鼠于阴道涂片见到大量角化上皮细胞的次日、其余各组剩余小鼠于注射HCG16h后，用摘除眼球法取血，留取血清后测定FSH、LH、E2的含量；然后脱颈椎处死小鼠，留取卵母细胞，计数卵泡数量及优质卵泡数。
结果：（1）各组小鼠获卵数及优质卵泡数比较：与空白对照组比较，COH组及各治疗组获卵数增加（P＜0.05），各治疗组与COH组比较均无统计学意义（P＞0.05），各治疗组之间获卵数比较无统计学意义（P＞0.05）；各治疗组优质卵泡数高于空白对照组及COH组（P＜0.05），COH组低于空白对照组（P＜0.05）；高剂量组优质卵泡数均优于低剂量组（P＜0.05），补肾高剂量组与疏肝高剂量组的优质卵泡数比较无统计学意义（P＞0.05），补肾低剂量组与疏肝低剂量组比较无统计学意义（P＞0.05）。（2）各组小鼠妊娠率及子宫胚胎数比较：COH组小鼠妊娠率较空白对照组降低（P＜0.05），补肾高剂量组和疏肝高剂量组妊娠率较空白对照组及COH组升高（P＜0.05），低剂量组与空白对照组比较无统计学意义（P＞0.05）；高剂量组小鼠妊娠率均优于低剂量组（P＜0.05），高剂量组间比较无统计学意义（P＞0.05），低剂量组间比较无统计学意义（P＞0.05）；各组之间子宫胚胎数比较均无统计学意义（P＞0.05）。（3）各组小鼠血清FSH、LH、E2值比较：与空白对照组比较，各治疗组及COH组血清FSH、LH、E2水平增高（P＜0.05）；与COH组比较，各治疗组血清FSH、LH、E2水平增高（P＜0.05），其中高剂量组的表达优于低剂量组（P＜0.05），补肾高剂量组与疏肝高剂量组之间无统计学意义（P＞0.05），补肾低剂量组与疏肝低剂量组之间无统计学意义（P＞0.05）。
结论：在用促性腺激素处理小鼠的同时加用补肾调经方和逍遥丸不会降低获卵数，并能增加优质卵母细胞数量，提高妊娠率及总胚胎数。
第二部分补肾法、疏肝法对小鼠卵母细胞GDF-9及Smads通路影响的比较
目的：通过研究补肾法、疏肝法对小鼠卵母细胞中卵母细胞分泌因子GDF-9、相关受体BMPRII、ALK-5及下游Smads通路中Smad2、Smad3的影响，探讨两法对卵母细胞质量影响的靶向性机制。
方法：选用6-7周龄健康雌性昆明小鼠600只，按随机数字表法完全随机化分为6组，即补肾高剂量组、补肾低剂量组、疏肝高剂量组、疏肝低剂量组、COH组和空白对照组，每组100只。补肾高、低剂量组分别给予补肾调经方口服液，疏肝高、低剂量组分别给予逍遥丸混悬液，COH组和空白对照组用蒸馏水灌胃。每日8:00各组小鼠均以1ml/100g体重灌胃，连续10天。补肾高剂量组、补肾低剂量组、疏肝高剂量组、疏肝低剂量组及COH组于第11日同时腹腔注射PMSG10IU/只，48h后腹腔注射HCG10IU/只；空白对照组每日阴道涂片，观察动情周期。注射HCG16h后，取补肾高低剂量组、疏肝高低剂量组和COH组小鼠，以及空白对照组阴道涂片见到大量角化上皮细胞的次日小鼠，脱颈椎处死，在体视镜下用1ml注射针划破输卵管壶腹部，收集卵丘卵母细胞复合体(COCs)，用透明质酸酶消化掉卵丘颗粒细胞后用PBS反复冲洗，获得MⅡ期卵母细胞，采用免疫组化法、实时荧光定量PCR分别检测卵母细胞中GDF-9、ALK-5、BMPR II、Smad2、Smad3蛋白及其mRNA表达；采用Western blot方法检测卵母细胞中GDF-9和BMPR II蛋白的表达。
结果：（1）与空白对照组比较，COH组GDF-9蛋白及mRNA的表达降低（P＜0.05），各治疗组的表达升高（P＜0.05）；高剂量组GDF-9蛋白及mRNA表达高于低剂量组（P＜0.05），补肾高剂量组与疏肝高剂量组比较无统计学意义（P＞0.05），补肾低剂量组与疏肝低剂量组比较无统计学意义（P＞0.05）。（2）与空白对照组比较，COH组、补肾高剂量组、补肾低剂量组及疏肝低剂量组BMPR II蛋白及mRNA表达无统计学意义（P＞0.05）；疏肝高剂量组BMPR II蛋白和mRNA的表达较其余各组均增强，有统计学意义（P＜0.05）。（3）ALK-5、Smad2、Smad3蛋白表达在各组间比较均无统计学意义（P＞0.05）。
结论：补肾调经方和逍遥丸均可以提高小鼠卵母细胞GDF-9蛋白及mRNA的表达，从而改善卵泡发育，提高卵母细胞质量，但其机制与GDF-9Ⅰ型受体及下游Smads通路无关。
第三部分补肾法、疏肝法对小鼠卵母细胞BMP-6及Smads通路影响的比较
目的：通过观察补肾法、疏肝法对小鼠卵母细胞中卵母细胞分泌因子BMP-6、相关受体ALK-2、ALK-6及下游Smads通路中Smad1、Smad5、Smad8和Smad4的影响，探讨两法对卵母细胞质量的影响的靶向性机制。
方法：动物分组及取材同第二部分。免疫组化和Western blot方法检测卵母细胞BMP-6、ALK-2、ALK-6、Smad1、Smad5、Smad8、Smad4蛋白的表达，实时荧光定量PCR法检测卵母细胞上述指标mRNA的表达。
结果：（1）BMP-6：与空白对照组比较，COH组卵母细胞BMP-6蛋白和mRNA的表达无统计学意义（P＞0.05），各治疗组表达升高，并高于COH组（P＜0.05）；疏肝高剂量组的表达高于补肾低剂量组和疏肝低剂量组（P＜0.05），低于补肾高剂量组（P＜0.05）；补肾低剂量组与疏肝低剂量组之间比较无统计学意义（P＞0.05）。（2）ALK-2：与空白对照组比较，COH组ALK-2蛋白及mRNA表达无统计学意义（P＞0.05）；各治疗组ALK-2的蛋白及mRNA表达均高于空白对照组和COH组（P＜0.05）；组间比较显示：各治疗组高剂量组ALK-2蛋白及mRNA表达均高于低剂量组（P＜0.05）；疏肝高剂量组表达较补肾高剂量组降低（P＜0.05），补肾低剂量组与疏肝低剂量组比较则无统计学意义（P＞0.05）。（3）ALK-6：与空白对照组比较，COH组ALK-6蛋白及mRNA表达无统计学意义（P＞0.05）；空白对照组和COH组与治疗组比较，ALK-6蛋白及mRNA表达降低（P＜0.05）；补肾组表达高于疏肝组（P＜0.05），补肾低剂量组的ALK-6蛋白及mRNA表达较补肾高剂量组降低（P＜0.05），疏肝高剂量组蛋白表达高于疏肝低剂量组（P＜0.05）。（4）Smad1：空白对照组与COH组比较无统计学意义（P＞0.05）；各治疗组Smad1蛋白及mRNA表达高于空白对照组和COH组（P＜0.05）；与补肾高剂量组比较，其余三个治疗组的表达降低（P＜0.05），补肾低剂量组较疏肝组蛋白及基因表达增强（P＜0.05），疏肝高剂量组Smad1蛋白及基因表达较疏肝低剂量组增强（P＜0.05）。（5）Smad5：与空白对照组比较，COH组表达无统计学意义（P＞0.05），各治疗组蛋白及mRNA表达增加，并高于COH组（P＜0.05）；高剂量组表达较低剂量组升高（P＜0.05），补肾高剂量组的表达高于疏肝高剂量组（P＜0.05），疏肝低剂量组较补肾低剂量组表达降低（P＜0.05）。（6）Smad8：空白对照组与COH组的Smad8蛋白及mRNA表达无统计学意义（P＞0.05）；各治疗组Smad8蛋白及mRNA表达较空白对照组和COH组升高（P＜0.05）；与高剂量组比较，各低剂量组的Smad8蛋白及mRNA表达均降低（P＜0.05），补肾高剂量组与疏肝高剂量组之间比较无统计学意义（P＞0.05），补肾低剂量表达较疏肝低剂量组增强（P＜0.05）。（7）Smad4：与空白对照组比较，COH组蛋白及mRNA表达无统计学意义（P＞0.05），各治疗组表达增高，有统计学意义（P＜0.05）；高剂量组Smad4蛋白及基因表达高于低剂量组（P＜0.05），补肾高剂量组表达高于疏肝高剂量组（P＜0.05），补肾低剂量组与疏肝低剂量组比较无统计学意义（P＞0.05）。
结论：逍遥丸可能通过提高小鼠卵母细胞BMP-6的表达，募集其Ⅱ型受体BMPRⅡ并激活ALK-2/6受体，启动下游Smad通路的信号传导；补肾调经方可能是通过募集其他Ⅱ型受体并激活ALK-2/6受体，启动下游Smad通路的信号传导；二者的作用机制有所差异。
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 outcomes
Objective: 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 oocytes
Objective: 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 oocytes
Objective: 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.
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