Regulatory Mechanism of Drug-Containing Serum of Jinghou Zengzhi Prescription on GDF9 Expression and Apoptosis of Ovarian Granulosa Cells in Rats with Controlled Ovarian Hyperstimulation / 广州中医药大学学报

Objective To observe the regulatory mechanism of drug-containing serum of Jinghou Zengzhi Prescription based on qi and blood replenishing method on the expression of growth and differentiation factor 9(GDF9)and apoptosis of ovarian granulosa cells in rats with controlled ovarian hyperstimulation(COH).Methods Serum of COH rats(blank serum)and serum of COH rats gavaged by the Jinghou Zengzhi Prescription were prepared.A COH rat model was established and ovarian granulosa cells were collected.The experiment was divided into 5 groups:blank serum group,drug-containing serum group,drug-containing serum+SB203580[p38 mitogen-activated protein kinase(p38MAPK)inhibitor]group,drug-containing serum + PDTC[nuclear transcription factor κB(NF-κB)inhibitor]group,drug-containing serum + SB203580 + PDTC group.The mRNA expression levels of p38MAPK,casein kinase 2(CK2),nuclear transcription factor κB inhibitor α(IκBα),NF-κB and GDF9 were detected by real-time quantitative polymerase chain reaction(qRT-PCR),and GDF9 protein expression level was detected by Western Blot,and ovarian granulosa cell apoptosis was detected by terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL).Results The drug-containing serum of Jinghou Zengzhi Prescription decreased the mRNA expressions of p38MAPK and NF-κB,elevated the mRNA expressions of CK2 and IκBα,increased the mRNA and protein expression levels of GDF9,and decreased the apoptosis rate of ovarian granulosa cells in COH rats.The addition of p38MAPK inhibitor SB203580 alone and the addition of NF-κB inhibitor PDTC alone both promoted the mRNA and protein expressions of GDF9 and reduced the apoptosis rate of granulosa cells.Conclusion The drug-containing serum of Jinghou Zengzhi Prescription based on qi and blood replenishing method can promote the expression of GDF9 and inhibit the apoptosis of ovarian granulosa cells in rats with COH,and its mechanism may be related to the regulation of the expression of genes of the dual signaling pathways of p38MAPK and NF-κB.

Clinical Observation on the Effect of Postmenstrual Proliferative Prescription Through Replenishing Qiand Blood on Ovulation Induction in Patients with Polycystic Ovarian Syndrome / 广州中医药大学学报

Objective To observe the effects of Postmenstrual Proliferative Prescription(mainly composed of Codonopsis Radix,Atractylodis Macrocephalae Rhizoma,Poria,Rehmanniae Radix Praeparata,Paeoniae Radix Alba,Angelicae Sinensis Radix,Chuanxiong Rhizoma,Cervi Cornu Degelatinatum,Corni Fructus,Cuscutae Semen,and Eucommiae Cortex)through replenishing qi and blood on the ovulation rate and pregnancy rate in patients with ovulatory dysfunction infertility caused by polycystic ovarian syndrome(PCOS)during ovulation-induction treatment,and to explore the therapeutic effects and possible therapeutic mechanism.Methods Sixty patients with ovulatory dysfunction infertility due to PCOS were randomly divided into a treatment group and a control group,with 30 patients in each group.The control group was given Clomifene Citrate Capsules to promote ovulation,and the treatment group was given Postmenstrual Proliferative Prescription on the basis of the ovulation-induction program of the control group starting from the fifth day of menstruation or progesterone withdrawal bleeding.The two groups were treated for one menstrual cycle as a course of treatment.The changes in the serum sex hormones of estradiol(E2),follicle-stimulating hormone(FSH),luteinizing hormone(LH),and progesterone(P)on the 2nd to 5th day of menstruation,as well as the changes in serum growth differentiation factor 9(GDF9)and bone morphogenetic protein 15(BMP15)levels on the 2nd to 5th day of menstruation and on the day of human chorionic gonadotropin(HCG)injection were observed in the two groups.Moreover,the ovulation rate,pregnancy rate and clinical efficacy of the patients in the two groups were analyzed.Results(1)No statistically significant differences in serum levels of sex hormones E2,FSH,LH and P on the 2nd to 5th day of menstruation were shown between the two groups of patients(P＞0.05).(2)On the 2nd to 5th day of menstruation and on the day of HCG injection,there were no significant differences in the serum GDF9 and BMP15 levels between the two groups(P＞0.05).(3)The ovulation rate and pregnancy rate in the treatment group were 93.33%(28/30)and 26.67%(8/30)respectively,which were significantly higher than 70.00%(21/30)and 13.33%(4/30)in the control group.And the differences tested by chi-square test were statistically significant between the two groups(P＜0.05).(4)The total effective rate of the treatment group was 93.33%(28/30),and that of the control group was 70.00%(21/30).The intergroup comparison(tested by rank sum test)showed that the therapeutic efficacy of the treatment group was significantly superior to that of the control group,and the difference was statistically significant(P＜0.05).Conclusion Postmenstrual Proliferative Prescription through replenishing qi and blood can improve the ovulation rate and pregnancy rate during ovulation-induction treatment in the patients with ovulatory dysfunction infertility due to PCOS.It is indicated that Postmenstrual Proliferative Prescription can enhance the quality of the oocytes and the potential of embryo implantation during the ovulation-induction treatment.

Effect of Jinghou Zengzhi Granules on ovarian GDF9 secretion and granulosa cells apoptosis in controlled ovarian hyperstimulation rats / 实用医学杂志

Objective To explore the effects of Jinghou Zengzhi Granules(JHZZG),on ovarian GDF9 secretion and granulosa cells(GCs)apoptosis in controlled ovarian hyperstimulation(COH)rats through p38MAPK/CK2/IκBα/NF-κB pathway.Methods A model of COH rats was prepared and 18 rats were randomly divided into 3 groups,including natural ovulation group(NO group),COH group,and COH+JHZZG group.The expression of p38MAPK,CK2,IκBα,NF-κB,GDF9 mRNA and protein were detected by qRT-PCR and Western blot,and the apoptosis rate of ovarian GCs by TUNEL.Results Compared with the NO group,the expression of p38MAPK and NF-κB in the ovarian tissue of rats in the COH group increased,while the expression of CK2,IκBα and GDF9 decreased,and the apoptosis rate of ovarian GCs increased(all P<0.01).Compared with the COH group,the expression of p38MAPK and NF-κB in the ovarian tissue of rats in the COH+JHZZG group decreased,while the expression of CK2,IκBα and GDF9 increased(all P<0.01),and the apoptosis rate of ovarian GCs decreased(P<0.05).Conclusion Jinghou Zengzhi Granules could promote the secretion of ovarian GDF9 and inhibit the apoptosis of ovarian GCs of COH rats through p38MAPK/CK2/IκBα/NF-κB signaling pathway,which thereby improve the quality of COH oocytes.

Single base editing system mediates site-directed mutagenesis of genes GDF9 and FecB in Ouler Tibetan sheep / 生物工程学报

In this study, a single base editing system was used to edit the FecB and GDF9 gene to achieve a targeted site mutation from A to G and from C to T in Ouler Tibetan sheep fibroblasts, and to test its editing efficiency. Firstly, we designed and synthesized sgRNA sequences targeting FecB and GDF9 genes of Ouler Tibetan sheep, followed by connection to epi-ABEmax and epi-BE4max plasmids to construct vectors and electrotransfer into Ouler Tibetan sheep fibroblasts. Finally, Sanger sequencing was performed to identify the target point mutation of FecB and GDF9 genes positive cells. T-A cloning was used to estimate the editing efficiency of the single base editing system. We obtained gRNA targeting FecB and GDF9 genes and constructed the vector aiming at mutating single base of FecB and GDF9 genes in Ouler Tibetan sheep. The editing efficiency for the target site of FecB gene was 39.13%, whereas the editing efficiency for the target sites (G260, G721 and G1184) of GDF9 gene were 10.52%, 26.67% and 8.00%, respectively. Achieving single base mutation in FecB and GDF9 genes may facilitate improving the reproduction traits of Ouler Tibetan sheep with multifetal lambs.

Growth and Differentiation Factor-9 Supplementation Affects Viability and Morphology of Preantral Follicles in Equine Ovarian Fragments During Short-term in vitro Culture

Abstract This study aimed to evaluate different concentrations of growth and differentiation factor-9 (GDF-9) on the development and maintenance of equine preantral follicle morphology during short-term in vitro culture. Ovaries (n=5) from five mares were collected from a local slaughterhouse and transported to the laboratory, where nine fragments (5x5x1mm) were procured from each ovary. One fragment from each was immediately fixed and submitted for histological analysis (control group; D0). The other eight fragments were cultured in situ for two (D2) or six (D6) days in MEM+ or MEM+ supplemented with GDF-9 at different concentrations (i.e., 50, 100 and 200 ng/mL the GDF-9). After culturing with different concentrations of GDF-9 for 2 or 6 days, the fragments were processed for histological analysis. After two days of cultivation, we observed an increase in the percentage of developing follicles for 0 (MEM+), 50, 100 and 200 ng/mL GDF-9 compared to control (D0; P<0.05). When we evaluated all treatments that preserved follicular integrity, the GDF-9 concentration of 100 ng/mL presented results superior to those of the other cultures (P<0.05). While, at six days of culture, the concentration of 200 ng/mL of GDF-9 appeared to be more efficient in providing development compared to MEM+ (P<0.05). The percentage of morphologically intact follicles in the 6 days culture samples treated with 50 ng/mL of GDF-9 indicated that this concentration was effective in maintaining the integrity of the follicle (P<0.05). We conclude, therefore, that graduated GDF-9 addition to the medium ensure follicular development and is sufficient maintain the architecture.

Effects of GDF-9 and FSH on mRNA expression for FSH-R, GDF-9 and BMPs in in vitro cultured goat preantral follicles

The aim of the present study was to determine the role of GDF-9 and/or FSH on the growth and mRNA expression for FSH-R, GDF-9, and BMPs in goat secondary follicles after culture in vitro. Goat secondary follicles (~200µm) were isolated and cultured for six days in minimum essential medium (MEM) supplemented with GDF-9 (200 ng/mL), FSH (50 ng/mL) or both. At the beginning and end of culture, the follicular diameter was evaluated and compared. The levels of mRNA for GDF-9, FSH-R and BMPs -2, -4, -6, -7 and -15 in cultured follicles were quantified by real time PCR. The results showed that a significant increase of follicle diameter after six days when compared to day 0, but the presence of GDF-9 and FSH did not influence the follicular growth in comparison with those cultured in MEM. Real time PCR showed that GDF-9 down-regulated the levels of mRNA for BMPs -2 and -15, while FSH either alone or in combination with GDF-9 did not affect the expression of GDF-9, FSH-R and BMPs. In conclusion, GDF-9 reduced the expression of BMP-2 and -15 in caprine preantral follicles after their culture, but FSH either alone or in association with GDF-9 did not control the expression of GDF-9, FSH-R and BMPs.

Growth differentiation factor 9 and bone morphogenetic protein 15 expression in previtellogenic oocytes and during early embryonic development of Yellow-tail Kingfish Seriola lalandi

BACKGROUND: During fish oocyte maturation, specific molecules are expressed and accumulated within oocyte until fertilization and embryo development. Special attention have been paid in members of the transforming growth factor (TGF-ß) superfamily; growth differentiation factor 9 (GDF9/gdf9) and bone morphogenetic protein 15 (BMP15/bmp15), which exert regulatory functions during oocyte maturation and follicle development. However, little attention has been paid to the involvement of these molecules during embryogenesis considering its importance for the formation of a good quality egg and subsequent embryo survival. The purpose of this study was to analyze the expression of gdf9 andbmp15 in previtellogenic oocytes and during early embryonic development in Seriola lalandi, a pelagic fish with increasing prospect for its aquaculture development, which however, show high mortality at embryo and larval stages. RESULTS: Through RT-qPCR it was found that gdf9 expression was higher in previtellogenic oocytes decreasing after ovulation. This expression profile agrees with its participation in early stages of the follicular development. The transcripts for bmp15 also showed the highest levels in previtellogenic oocytes, however this expression was lower than obtained with gdf9. Conversely, in recently spawned oocytes mRNA bmp15 levels were highest than observed to gdf9. This, is consequent with the main role proposed for this growth factor at the final fish oocyte maturation: avoid the ovulation of an immature oocyte. During embryo development, low levels of mRNA were detected to gdf9, with an increase in 48 H post-fertilization embryos. The bmp15 expression did not change throughout development and was higher than gdf9 at 16 cells, blastula and appearance embryos stages. CONCLUSIONS: Both (gdf9 and bmp15) expression profiles in previtellogenic oocytes and newly spawned eggs are consistent with the described functions for these growth factors in vertebrate ovarian physiology in early and late stages of the follicular development. So, these genes could be considered as quality biomarkers at these stages. However, further studies of these proteins throughout folliculogenesis, are necessaries to fully understand their functions during the oocyte formation. In addition, the persistent expression of these growth factors during development, allows us to speculate possible roles in embryonic processes, which must also be addressed.

Interaction between growth differentiation factor 9, insulin-like growth factor I and growth hormone on the in vitro development and survival of goat preantral follicles

The objective of this study was to determine the effects of GDF-9, IGF-I, and GH alone or combined on preantral follicle survival, activation and development after 1 and 7 days of in vitro culture. Either fresh (non-cultured) or cultured ovarian tissue was processed for histological and fluorescence analysis. For all media tested, the percent of normal follicles was greater when compared to minimum essential medium supplemented (MEM+) alone, except when ovarian tissue was cultured with GDF-9/IGF-I or GDF-9/GH (P < 0.05). Fluorescence analysis showed that the percent of viable follicles after 7 days of culture was similar for non-cultured tissue and for all treatments tested. The percent of primordial follicles was reduced (P < 0.05) and there was a significant and concomitant increase in the percent of intermediate and primary follicles in all treatments tested after 7 days of culture when compared to non-cultured tissue. After 7 days of culture, the highest percent of intermediate follicles was observed with IGF-I/GH (61.3 percent), and the highest percent of primary follicles was achieved with IGF-I (57.7 percent). After 7 days of culture in MEM+ containing GDF-9, IGF-I and GH alone or in all associations, a significant increase in follicular diameter was observed when compared to MEM+ alone and non-cultured tissue. In conclusion, GDF-9, IGF-I and GH alone or in combination maintain preantral follicle survival and promote primordial follicle activation. Nevertheless, the data showed that IGF-I/GH and IGF-I alone are efficient in promoting the transition from primordial to intermediate follicles and from intermediate to primary follicles, respectively.

mRNA Expression of the Regulatory Factors for the Early Folliculogenesis in vitro / 대한불임학회지

OBJECTIVE: To understand the crucial requirement for the normal early folliculogenesis, we evaluated molecular as well as physiological differences during in vitro ovarian culture. Among the important regulators for follicle development, anti-Mullerian hormone (AMH) and FSH Receptor (FSHR) have been known to be expressed in the cuboidal granulosa cells. Meanwhile, it is known that c-kit is germ cell-specific and GDF-9 is also oocyte-specific regulator. To evaluate the functional requirement for the competence of normal follicular development, we investigated the differential mRNA expression of several factors secreted from granulosa cells and oocytes between in vivo and in vitro developed ovaries. MATERIALS AND METHODS: Ovaries from ICR neonates (the day of birth) were cultured for 4 days (for primordial to primary transition) or 8 days (for secondary follicle formation) in alpha-MEM glutamax supplemented with 3 mg/ml BSA without serum or growth factors. The mRNA levels of the several factors were investigated by quantitative real-time PCR analysis. Freshly isolated 0-, 4-, and 8-day-old ovaries were used as control. RESULTS: The mRNA of AMH and FSHR as granulosa cell factors was highly increased according to the ovarian development in both of 4- and 8-day-old control. However, the mRNA expression was not induced in both of 4- and 8-day in vitro cultured ovaries. The mRNA expression of GDF-9 known to regulate follicle growth as an oocyte factor was different between in vivo and in vitro developed ovaries. In addition, the transcript of GDF-9 was expressed in the primordial follicles of mouse ovaries. The mRNA expression of c-kit was not significantly different during the early folliculogenesis in vitro. CONCLUSION: This is the first report regarding endogenous AMH and FSHR expression during the early folliculogenesis in vitro. In conclusion, it will be very valuable to evaluate cuboidal granulosa cell factors as functional marker(s) for normal early folliculogenesis in vitro.

Mutational Analysis of Growth Differentiation Factor-9 Gene in Korean Women with Premature Ovarian Failure / 대한산부인과학회잡지

The clinical models for studying ovary-determining genes may be premature ovarian failure (POF). POF is a condition causing amenorrhea, hypoestrogenism, and elevated gonadotropins in women under 40 years old. FSH receptor, LH receptor, inhibin, GDF-9 (growth differentiation factor-9), BMP-15 (bone morphogenetic protein-15), DIAPH2 (diaphanous gene) and XPNPEP2 (X-prolyl aminopeptidase) genes were proposed as a possible candidate gene, but until recently, only mutations in FSH receptor, LH receptor and inhibin genes have been identified in POF patients. Therefore mutation screening of another POF gene necessary to reveal the principal causative genes of POF. OBJECTIVE: The present study was performed to analyze the mutation of GDF-9 gene in Korean patient with POF and to investigate whether mutation of these gene is a likely main cause of POF. METHODS: Eighty-six women with POF were studied and thirty-six normal women were enrolled as control. Mutation screening of these genes were performed by denaturing HPLC and were confirmed by automatic sequencing. RESULTS: Three different mutations of GDF-9 gene were identified in Korean women with POF; Arg3Cys mutation in one patient, Leu40Val mutation in one patient, Asp57Tyr mutation in 10 patients and 5 normal controls. Arg3Cys mutation and Leu40Val mutation were likely cause of disease. Frequencies of Arg3Cys mutation and Leu40Val mutation were 1.2%, respectively. Asp57Tyr mutation was common polymorphism in Korean women. All mutations was a novel mutation found in the present study. CONCLUSION: POF was resulted by mutations of GDF-9 gene, but mutations of GDF-9 gene are not likely main causes of POF because of low frequency of mutations.