Comparative Study Of Bushen Treatment And Shugan Treatment Regulating BMP15/BMP6-smads-sohlh2 Signal Pathway In Mice Oocytes Of Preantral Follicles Cultured In Vitro

Ovary performs both a reproduction and endocrine function, and ovary dysfunction is an important reason for women’s weak reproductive capacity.The incidence of infertility has ran up to 10%~15% recently in China, which has been increasing year after year under both social and iatrogenic factors,and influences women’s health and family happiness seriously. At present,assisted reproductive technology(ART), whose representative is in vitro fertilization-embryo transfer technology(IVF-ET), is one of the important means to solve the problem of infertility, while it is still difficult to get rid of the high abortion rate and low pregnancy rate for the lack of high-quality oocyte. Preantral follicle is the basic unit in the ovary of mammal, including human, which plays an important role in maintaining female’s reproductive life. Although the number of preantral follicles is numerous, but more than99% of them undergo an involutional process called follicular atresia with ontogenetic development, which undoubtedly is a colossal waste of reproductive resources. Therefore, a good knowledge of the mechanism of preantral follicular growth and a full utilization of the resource have far-reaching and meaningful medical value.The quality of oocyte is affected by multiple factors. With the rapid development of molecular biology and genetic engineering techniques, the knowledge of the regulatory mechanisms of follicular development is deepened. The local multi-factors regulating system in ovary especially the oocyte-secreted factors(OSFs) have become the focus of attention of reproductive researchers, which form a huge challenge to hypothalamicpituitary-ovarian axis regulatory theory. Bone morphogenetic protein 15(BMP15) and bone morphogenetic protein 6(BMP6) both are importantmembers of OSFs, playing an important role in follicular growth, dominant follicles selection, cumulus expansion, ovulation, luteal formation, follicular atresia and so on. Drosophila mothers against decapentaplegic proteins(Smads) signal pathway is the main way for them to function properly. Typing with Ⅱ receptor and Ⅰ receptor is the first, and then receptor-regulated Smads in cell will be phosphorylated by activated Ⅰ receptor, at last the phosphorylated Smads are transferred to nuclear with the help of common-mediator Smad to regulate the transcription of relative gene.In recent years, traditional Chinese medicine(TCM) has played an increasingly prominent role in promoting follicular development and improving oocyte quality. Our research team studied the effects of Bushen Tiaojing recipe(BTR) and Xiaoyao powder on OSFs in granulosa cells and follicular fluid of IVF-ET patients and the outcome of pregnancy, and conclued that they both can increase the number of oocyte and high quality embryo rate, at the same time improve clinical pregnancy rate by different means, Bushen treatment through up-regulating the expression of BMP15 and BMP6 in granulosa cells as well as BMP6 in follicular fluid, while Shugan treatment by up-regulating the expression of BMP15 in granulosa cells only.Futhermore a comparative study on the effects of BTR and Xiaoyao Powder/Pill(XYP) on OSFs-Smads signal pathway in granular cells of IVF-ET patients and oocytes of controlled ovarian hyperstimulation mice was carried out later and a conclusion that the possible mechanism for Bushen treatment and Shugan treatment improving the quality of oocytes was related to BMP15/BMP6-Smads signal pathway, and the specific points were not completely the same was reached. But a deeper discussion on the target gene of Smads signal pathway in oocyte of preantral follicle cultured in vitro was not involved. Primary cell culturing could preserve the intrinsic characteristics maximumly, and preantral follicle cultured in vitro is a good model for mechanism research of TCM. Based on the previous research, in this study,the effects of serum containing BTR or XYP on the main effector molecules of BMP15/BMP6-Smads-sohlh2 signal pathway in mice oocytes of preantralfollicles cultured in vitro were observed, in order to explore the possible target gene of BMP15/BMP6-Smads signal pathway, explain the potential molecular mechanism of Bushen treatment and Shugan treatment improving the quality of oocytes of preantral follicles cultured in vitro, find out the same and different points between them, thus to improve reproductive theory of TCM and supply scientific basis for follicle culture system optimization and ART further development.Part one Comparison of the effects of Bushen treatment and Shugan treatment on the survival rate and diameters of mice preantral follicles cultured in vitroObjective: To explore the regulation function of BTR and XYP on mice preantral follicles cultured in vitro by observing the effects of serum containing them on the survival rate and diameters of the follicles on D6.Methods:Preparation for rat serum containing BTR or XYP28 female 6-week-old SD rats were randomly distributed into seven groups: low, middle, high dose Bushen groups, low, middle, high dose Shugan groups and blank group(N=4). Respectively they were drenched with1.54g/ml, 3.08g/ml, 4.62g/ml BTR enriched oral liquid, 0.09g/ml, 0.18g/ml,0.27g/ml XYP suspension or distilled water with the dose of 1ml/(100g·d)twice a day for three days. And on D4, after fasted for 12 hours they were drenched with the whole day dose one time. After 1 hour they were anesthetized with 10% chloral hydrate and the blood was drawed from femoral artery. The blood was centrifuged at 3000 revolution per minute for 15 minutes at room temperature after taken into 37℃ water bath for 15 minutes.Then the supernatant was sucked, and put into 56℃water bath for 30 minutes.Finally the serum from the same group was mixed, filtered for sterilization and subpacked. Thus rat serum containning different concentrations of BTR(low,middle, high dose), XYP(low, middle, high dose) and normal serum was gotten for the following experiment.Preantral follicles isolationThe preantral follicles were microisolated mechanically. 24 12-day-old Kunming mice were killed under bacteria-free environment by the cervical dislocation method and their ovaries were taken out quickly and washed with saline containing 200IU/ml penicillin and 200μg/ml streptomycin for 2 to 3times. Then they were put into the operating liquid and delivered to the super-clean bench immediately. Under an 40 times stereoscope, the ovaries were gashed and preantral follicles were isolated with 25 G needles quickly. Be careful not to damage the follicular basement membrane.Preantral follicles culturing grouply With improved common culture method, preantral follicles with the diameter between 80μm to 120μm and integrated morphological features were selected out to be incubated in a 35 mm culture vessel with culture solution balanced overnight beforehand, which was placed in a three gas incubator with saturated humidity and 5% CO2, at 37℃. Once the follicles stuck to the bottom of the culture dishes, they were supplemented with 10% normal rat serum, 10% rat serum containing differernt concentrations of BTR or XYP(low, middle, high dose) respectively, and then they were divided into the normal group(NG), Bushen low dose group(BL), Bushen middle dose group(BM), Bushen high dose group(BH), Shugan low dose group(SL), Shugan middle dose group(SM), and Shugan high dose group(SH). The group without rat serum was blank group(BG). All the follicles were incubated for 6days, and observed under inverted microscope everyday for the morphological changes. On D6, the survival number and diameters of follicles in each group were counted and measured.Results:1 the morphological changes of follicles in each group On D2, the follicles adhered to the bottom of the culture dishes and oocytes were clearly observed; On D4, granulosa cells significantly increased in quantity and the bottom of the culture dishes were covered with cells. On D6, the granulosa cells duplicated quickly and crossed over the follicular theca.At the same time, oocytes could not be seen clearly since they were veiled bythe granulosa cells.2 Comparison of the survival rate of follicles in each group There were no statistical significance on the survival rate of follicles among BG, NG, BL, BM, SL and SM(P>0.05). Compared with them, the survival rate in BH and SH increased(P<0.05), and that in BH was higher than SH(P<0.05).3 Comparison of the follicular diameter in each group Diameter in BG and NG showed no significant difference(P>0.05), and in the administration groups it increased(P<0.05). Among Bushen groups, the diameter in BH was the longest in a dose-dependent manner(P<0.05). Among Shugan groups, diameter in SH was longer than SM and SL(P<0.05), and there was no difference between SM and SL(P>0.05). Among administration groups, diameter in BH was longer than Shugan groups(P<0.05), in BM it was longer than SM and SL(P<0.05), and BL and SL showed no significant difference(P>0.05).Conclusion: Bushen treatment and Shugan treatment both can improve the survival rate and diameters of preantral follicles cultured in vitro to promote follicular development, futhermore Bushen treatment was better than Shugan treatment.Part two Comparison of the effects of Bushen treatment and Shugan treatment on BMP15 and BMP6 in mice oocytes during the development of preantral follicles cultured in vitroObjective: To explore the relationship between BTR and XYP improving the quality of oocytes during the development of preantral follicles cultured in vitro and regulating the expression of BMP15 and BMP6 by observing the effects of serum containing them on BMP15 and BMP6 expression in oocytes.Methods: Preantral follicles isolation and grouply culture are the same to those in part one. Oocytes collection: Enzyme digesting method was adopted.Respectively on D2, D4, D6 the preantral follicles were suspended under stereomicroscope, digested with 0.25% collagenase NB4 and 0.1%hyaluronidase for 5 to 10 minutes at 37℃ in turns after centrifugation, andpipetted gently during the procedure. And the oocytes were collected after rinsed repeatedly with PBS. BMP15, BMP6 m RNA and protein expression in oocytes were respectively detected by real-time fluorescence quantitative polymerase chain reaction(real-time PCR), celluar immunofluorescence(IF)along with cytometric bead array(CBA).Results:1 Comparison of BMP15, BMP6 m RNA and protein expression in oocytes of each group in vitro on D2 Compared with BG, BMP15, BMP6 m RNA and protein expression in NG showed no statistically significant difference(P>0.05), and administration groups increased(P<0.05). Among Bushen groups, BMP15, BMP6 m RNA and protein in BH were the highest in a dose-dependent manner(P<0.05),while among Shugan groups, BMP6 protein in SH was higher than that in SM and SL(P<0.05), and there was no difference between SM and SL(P>0.05).Among administration groups, BMP15, BMP6 m RNA and protein expression in BH were higher than Shugan groups(P<0.05); BMP15 m RNA, BMP6 m RNA and protein in BM were higher than those in SM and SL(P<0.05),BMP15 protein in BM and SM showed no statistical difference(P>0.05), both higher than that in SL(P<0.05); BMP15 m RNA in BL was higher than that in SL(P<0.05), while BMP15 protein, BMP6 m RNA and protein showed no difference between the two groups(P>0.05).2 Comparison of BMP15, BMP6 m RNA and protein expression in oocytes of each group in vitro on D4 Compared with BG, BMP15, BMP6 m RNA and protein expression in NG showed no statistically significant difference(P>0.05), and administration groups increased(P<0.05). Among Bushen groups, BMP15, BMP6 m RNA and protein in BH were the highest in a dose-dependent manner(P<0.05),while among Shugan groups, BMP6 m RNA and protein in SH were higher than those in SM and SL(P<0.05), and there was no difference between SM and SL(P>0.05). Among administration groups, BMP15, BMP6 m RNA and protein expression in BH were higher than Shugan groups(P<0.05); BMP15m RNA and protein in BM, higher than those in SL(P<0.05), showed no statistical difference with SM(P>0.05), while BMP6 m RNA and protein in BM were higher than those in SM and SL(P<0.05); BMP15 m RNA in BL was higher than SL(P<0.05), while BMP15 protein, BMP6 m RNA and protein showed no difference between the two groups(P>0.05).3 Comparison of BMP15, BMP6 m RNA and protein expression in oocytes of each group in vitro on D6 Compared with BG, BMP15 and BMP6 m RNA and protein expression in NG showed no statistically significant difference(P>0.05), and administration groups increased(P<0.05). Among Bushen groups, BMP15, BMP6 m RNA and protein in BH were the highest in a dose-dependent manner(P<0.05),while among Shugan groups, BMP15 m RNA and protein in SH, higher than SL, showed no difference with SM. Among administration groups, BMP15,BMP6 m RNA and protein expression in BH were higher than Shugan groups(P<0.05); BMP15, BMP6 m RNA and protein in BM were higher than those in SM and SL; BMP15 m RNA and protein in BL showed no statistical difference with SL(P<0.05), while BMP6 m RNA and protein in BL were higher than those in SL(P<0.05).4 Comparison of BMP15, BMP6 m RNA and protein expression in oocytes of each group at different time As to BMP15 m RNA and protein, the expression in BG and NG on D2,D4, D6 were gradually increased, but the difference was not significant(P>0.05); the expression in BL, BM, SL and SM on D2 and D4 showed no difference(P>0.05), both lower than D6(P<0.05); the expression in BH were gradually increased, while in SH D4 and D6, higher than D2(P<0.05),showed no difference(P>0.05). To BMP6 m RNA and protein, the results of BG and NG are the same to BMP15; the expression in Bushen groups were gradually increased(P<0.05); the expression in SL and SM on D2 and D4 showed no difference(P>0.05), both lower than D6(P<0.05); while in SH on D4 and D6 showed no difference(P>0.05), both higher than those on D2(P<0.05).Conclusion: Bushen treatment and Shugan treatment both could improve the oocytes quality, which may be relevant to up-regulating the expression of BMP15 and BMP6 m RNA and protein in oocytes during the development of preantral follicles in vitro. Futhermore the function was more significant as times going and Bushen treatment was better than Shugan treatment.Part three Comparison of the effects of Bushen treatment and Shugan treatment on the membrane receptors of BMP15 and BMP6, along with Smads in mice oocytes of preantral follicles cultured in vitroObjective: To explore the potential molecular mechanisms for BTR and XYP improving the quality of oocytes by observing the effects of serum containing them on ALK2, ALK6, BMPRⅡ, Smad1, Smad5, Smad8 and p-Smad1/5/8 in oocytes of preantral follicles cultured in vitro on D6.Methods: Preantral follicles isolation and culture method are the same to part one. Suitable preantral follicles were selected out and divided into 10 groups randomly: BG, NG, BL, BM, BH, Bushen inhibitor group(BI), SL,SM, SH and Shugan inhibitor group(SI). Except for BG, the others were supplemented with 10% normal rat serum, 10% rat serum containing differernt concentrations of BTR or XYP(low, middle, high, high dose + 10μmol/L K02288) respectively.Oocytes collection: BI and SI were supplemented with K02288 2 hours before collection, and the collection operation was the same to part two.ALK2, ALK6, BMPRⅡ, Smad1, Smad5, Smad8 m RNA were detected by real-time PCR, the above indexs and p-Smad1/5/8 protein expression were detected by IF, and ALK2, ALK6, BMPRⅡ, Smad1, Smad5 protein expression were detected by CBA, too.Results:1 Comparison of ALK2, ALK6 m RNA and protein expression in oocytes of each group Compared with BG, ALK2, ALK6 m RNA and protein expression in NG showed no statistically significant difference(P>0.05), and administration groups increased(P<0.05). Among Bushen groups, ALK2, ALK6 m RNA andprotein in BH were the highest in a dose-dependent manner(P<0.05), and Shugan groups were the same(P<0.05). Among administration groups, ALK2,ALK6 m RNA and protein expression in BH were higher than Shugan groups(P<0.05); ALK2, ALK6 m RNA and protein in BM were higher than SM and SL(P<0.05); ALK2 m RNA and protein in BL were higher than SL(P<0.05),while ALK6 m RNA and protein in BL showed no statistical difference with SL(P>0.05).2 Comparison of BMPRⅡ m RNA and protein expression in oocytes of each group Compared with BG, BMPRⅡ m RNA and protein expression in NG showed no statistically significant difference(P>0.05), and administration groups increased(P<0.05). Among Bushen groups, BMPRⅡ m RNA and protein in BH were the highest in a dose-dependent manner(P<0.05), and Shugan groups were the same(P<0.05). Among administration groups,BMPRⅡ m RNA and protein expression in SH were higher than Bushen groups(P<0.05); BMPRⅡ m RNA and protein in SM were higher than BM and BL(P<0.05); BMPRⅡ m RNA and protein in SL were higher than BL(P<0.05).3 Comparison of Smad1, Smad5, Smad m RNA and protein expression in oocytes of each group As to Smad1 m RNA and protein, there was no statistically significant difference among BG, NG, BL, SL, SM(P>0.05), and those in BM, BH, SH increased(P<0.05). Those in BH higher than BM(P<0.05), showed no difference with SH(P>0.05).Compared with BG, Smad5, Smad8 m RNA and protein expression in NG showed no statistically significant difference(P>0.05), and administration groups increased(P<0.05). Among Bushen groups, Smad5 m RNA and protein in BH were the highest in a dose-dependent manner(P<0.05), Smad8 m RNA and protein in BH, higher than BL(P<0.05), showed no difference with BM(P>0.05). Among Shugan groups,Smad5 m RNA and protein in SH higher than SL(P<0.05), showed no difference with SM(P>0.05), while Smad8mRNA and protein in SH were the highest in a dose-dependent manner(P<0.05). Among administration groups, Smad5 m RNA and protein in SH and BH showed no difference, both higher than BM and BL(P<0.05), those in SM were higher than BM and BL(P<0.05), and those in SL were higher than BL(P<0.05); Smad8 m RNA and protein expression in BH were higher than Shugan groups(P<0.05), in BM were higher than SM and SL(P<0.05), and in BL were higher than SL(P<0.05).4 Comparison of p-Smad1/5/8 protein expression in oocytes of each group Compared with BG, p-Smad1/5/8 protein expression in NG showed no statistically significant difference(P>0.05), and both Bushen groups and Shugan groups increased(P<0.05). Among Bushen groups, p-Smad1/5/8protein in BH was the highest in a dose-dependent manner(P<0.05), and Shugan groups was the same(P<0.05). Among Bushen groups and Shugan groups, p-Smad1/5/8 protein expression in BH was higher than Shugan groups(P<0.05); p-Smad1/5/8 protein in BM was higher than SM and SL(P<0.05);and p-Smad1/5/8 protein in BL was higher than SL(P<0.05). Compared with BH and SH, p-Smad1/5/8 protein expression in BI and SI decreased respectively(P<0.05).Conclusion: Bushen treatment and Shugan treatment could possibly improve the oocytes quality in vitro by increasing the expression of ALK2,ALK6, BMPRⅡ, Smad1, Smad5, Smad8 m RNA and protein, p-Smad1/5/8protein in oocytes, up-regulating phosphorylation function of Ⅰ receptor, and enhancing the BMP15/BMP6-Smads signal transduction. What is more,Bushen treatment was better than Shugan treatment on up-regulating the expression of type Ⅰ receptors, Smad1, Smad8 and p-Smad1/5/8, while weaker on up-regulating type Ⅱ receptor and Smad5 expression.Part four Comparison of the effects of Bushen treatment and Shugan treatment on sohlh2 in mice oocytes of preantral follicles cultured in vitroObjective: To explore the potential target of BTR and XYP improving the quality of oocytes by observing the effects of serum containing them onsohlh2 in oocytes of preantral follicles cultured in vitro on D6Methods: Preantral follicles isolation, culture method were the same to part one, and grouping, oocytes collection were the same to part three.Respectively sohlh2 m RNA and protein expression were detected by real-time PCR and IF.Results: Among BG, NG, BL, SL, SM, sohlh2 m RNA and protein expression showed no statistical difference(P>0.05), and in BM, BH, SH they increased(P<0.05). Followed with BM, those in BH were the highest(P<0.05). Respectively compared with BH and SH, p-Smad1/5/8 protein expression in BI and SI decreased(P<0.05).Conclusion: Sohlh2 may be the target gene of BTR and XYP regulating the BMP15/BMP6-Smads signal pathway, and on up-regulating sohlh2 expression Bush treatment was better than Shugan treatment.In summary, Bushen treatment and Shugan treatment could promote preantral follicular development in vitro and improve oocytes quality possibly by regulating the BMP15/BMP6-Smads-sohlh2 signal pathway in oocytes. On the one hand they could promote BMP15 and BMP6 to combine with BMPRII,and then respectively type with ALK6, ALK2 and ALK6. On the other hand,they could enhance the phosphorylation function of Ⅰ receptor. Finally the target gene sohlh2 was influenced. In a word, the BMP15/BMP6-Smads signal transduction was enhanced. Furthermore, on up-regulating the expression of BMP15, BMP6, ALK2, ALK6, Smad1, Smad8, p-Smad1/5/8 and sohlh2 Bushen treatment was better than Shugan treatment, while weaker on BMPRII and Smad5.