Decreased growth differentiation factor 9, bone morphogenetic protein 15, and forkhead box O3a expressions in the ovary via ulipristal acetate.

OBJECTIVE: Folliculogenesis is a complex process involving various ovarian paracrine factors. During folliculogenesis, vitamin D3 and progesterone are significant for the proper development of follicles. This study aimed to investigate the effects of vitamin D3 and selective progesterone receptor modulator ulipristal acetate on ovarian paracrine factors. METHODS: In the study, 18 female Wistar-albino rats were randomly divided into three groups: control group (saline administration, n=6), vitamin D3 group (300 ng/day vitamin D3 oral administration, n=6), and UPA group (3 mg/kg/day ulipristal acetate oral administration, n=6). Ovarian tissue was analyzed by histochemistry and immunohistochemistry. For quantification of immunohistochemistry, the mean intensities of growth differentiation factor 9, bone morphogenetic protein 15, and forkhead box O3a expressions were measured by Image J and MATLAB. Blood samples were collected for the analysis of serum anti-Müllerian hormone levels by ELISA. RESULTS: Atretic follicles and hemorrhagic cystic structures were observed in the UPA group. After immunohistochemistry via folliculogenesis assessment markers, growth differentiation factor 9, bone morphogenetic protein 15, and cytoplasmic forkhead box O3a expressions decreased in the UPA group (p<0.05). Anti-Müllerian hormone level did not differ significantly between the experimental groups (p>0.05). CONCLUSION: Ulipristal acetate negatively affects folliculogenesis via ovarian paracrine factors. The recommended dietary vitamin D3 supplementation in healthy cases did not cause a significant change.

Biochanin-A attenuates DHEA-induced polycystic ovary syndrome via upregulation of GDF9 and BMP15 signaling in vivo.

AIMS: Phytoestrogens can act as natural estrogens owing to their structural similarity to human estrogens. Biochanin-A (BCA) is a well-studied phytoestrogen with a wide variety of pharmacological activities, whereas not reported in the most frequently encountered endocrinopathy called polycystic ovary syndrome (PCOS) in women. PURPOSE: This study aimed to investigate the therapeutic effect of BCA on dehydroepiandrosterone (DHEA) induced PCOS in mice. MAIN METHODS: Thirty-six female C57BL6/J mice were divided into six groups: sesame oil, DHEA-induced PCOS, DHEA + BCA (10 mg/kg/day), DHEA + BCA (20 mg/kg/day), DHEA + BCA (40 mg/kg/day), and metformin (50 mg/kg/day). KEY FINDINGS: The results showed a decrease in obesity, elevated lipid parameters, restoration of hormonal imbalances (testosterone, progesterone, estradiol, adiponectin, insulin, luteinizing hormone, and follicle-stimulating hormone), estrus irregular cyclicity, and pathological changes in the ovary, fat pad, and liver. SIGNIFICANCE: In conclusion, BCA supplementation inhibited the over secretion of inflammatory cytokines (TNF-α, IL-6, and IL-1ß) and upregulated TGFß superfamily markers such as GDF9, BMP15, TGFßR1, and BMPR2 in the ovarian milieu of PCOS mice. Furthermore, BCA reversed insulin resistance by increasing circulating adiponectin levels through a negative correlation with insulin levels. Our results indicate that BCA attenuated DHEA-induced PCOS ovarian derangements, which could be mediated by the TGFß superfamily signaling pathway via GDF9 and BMP15 and associated receptors as first evidenced in this study.

Efficacy and Clinical Significance of the Zuogui Pill on Premature Ovarian Failure via the GDF-9/Smad2 Pathway.

Our study aims to investigate the efficacy and clinical significance of the Zuogui pill (ZGP) on premature ovarian failure (POF) via the GDF-9/Smad2 pathway. Changes in clinical symptoms in the control group (treated with Femoston alone) and the treatment group (treated with ZGP combined with Femoston) were assessed before and after treatment. Sex hormone levels, serum inflammatory cytokine levels, and ultrasound parameters were measured before and after treatment. POF rat models were established using cyclophosphamide and the POF rats were treated with Femoston, or ZGP combined with Femoston. GDF-9 and Smad2 expression levels were determined by RT-qPCR. The follicle-stimulating hormone (FSH), luteinizing hormone (LH), interleukin (IL)-6, and IL-21 levels, and the pulsatility index (PI) and resistance index (RI) values were decreased, while the estradiol (E2) and anti-Mullerian hormone (AMH) levels, antral follicle count (AFC), ovarian volume (OV), mean ovarian diameter (MOD), and peak systolic velocity (PSV) values were increased in the treatment group compared to the control group. After treatment with ZGP combined with Femoston, GDF-9 and Smad2 expression in the ovarian tissues of POF rats increased. ZGP has a therapeutic effect on POF via modulation of the GDF-9/Smad2 pathway.

L-Cysteine improves bovine oocyte developmental competence in vitro via activation of oocyte-derived growth factors BMP-15 and GDF-9.

This study was designed to investigate the effect of different concentrations of L-cysteine supplementation into the maturation medium on the oocyte nuclear maturation, cumulus cell expansion, ultrastructure of the oocytes and the expression of oocyte-derived growth factors BMP-15, GDF-9 and CB-1 genes. Cumulus oocyte complexes (COCs) were collected from cow's ovaries obtained from abattoir and incubated at 38.5°C in maturation media supplemented with 0, 0.6, 0.8 or 1 mM L-cysteine in 5% CO2 under humidified air for 24 hr. We found that a significantly higher percentage of oocytes progressed to metaphase II stage in the in vitro maturation (IVM) medium supplemented with L-cysteine, particularly 0.8 mM group, compared with untreated control oocytes. Additionally, L-cysteine treatment significantly increased the number of expanded COCs and the degree of expansion of individual COCs. Results of RT-qPCR showed significant increase in expression levels of BMP-15 and GDF-9 in L-cysteine-treated groups compared with control one. Electron microgram showed improvement of cytoplasmic maturation regarding ultrastructure of the oocytes and oocyte-cumulus cell gap junction communication in all L-cysteine-treated groups especially 0.8 mM L-cysteine-treated one. In conclusion, supplementation of IVM medium with a potential anti-oxidant, L-cysteine can effectively improve in vitro oocytes cytoplasmic and nuclear maturation via activation of oocyte maturation related BMP-15 and GDF-9 genes in bovine oocytes, benefiting the extended researches about the potential applications of L-cysteine in mammalian breeding technologies.

The boosting effects of melatonin on the expression of related genes to oocyte maturation and antioxidant pathways: a polycystic ovary syndrome- mouse model.

BACKGROUND: Melatonin, as a free radical scavenger exhibiting genomic actions, regulates the antioxidant genes expression and apoptosis mechanisms. In polycystic ovary syndrome (PCOS) patients, an imbalance between free radicals and antioxidants in follicular fluid leads to oxidative stress, aberrant folliculogenesis, and intrinsic defects in PCOS oocytes. In this experimental mouse model study, oocytes of PCOS and the control groups were cultured in different melatonin concentrations (10- 5, 10- 6, and 10- 7 M) to investigate the expression of oocyte maturation-related genes (Gdf9/Bmp15), antioxidant-related genes (Gpx1/Sod1), apoptotic biomarkers (Bcl2/Bax) and total intracellular ROS levels. RESULTS: Gdf9 and Bmp15, Gpx1 and Sod1 were up-regulated in PCOS and control oocytes cultured in all melatonin concentrations compared to those cultured in IVM basal medium (P < 0.05). A significant decrease in the total ROS level was observed in all groups cultured in the supplemented cultures. Melatonin increased Bcl2 and decreased Bax gene expression in PCOS and control oocytes compared to non-treated oocytes. CONCLUSIONS: Melatonin increased antioxidant gene expression and regulated the apoptosis pathway, effectively reducing the adverse effects of culture conditions on PCOS oocytes. Furthermore, it influenced the expression of oocyte maturation-related genes in PCOS, providing valuable support during the IVM process.

The effect of Kuntai capsule on ovarian function in cisplatin-induced premature ovarian insufficiency rats.

Objective: This study aims to evaluate the effect of Kuntai capsule on ovarian function in cisplatin-induced premature ovarian insufficiency rats and to explore the mechanism of Kuntai capsule on the ovarian function of rats. Methods: Seventy-four female Sprague-Dawley rats were used for this study. Eight of the rats were randomly assigned to the Control group. The remaining sixty-six rats were utilized to establish the POI model via Cisplatin and then randomly divided into four groups: the model Control group, the Estradiol group, and groups treated with low and high doses of Kuntai capsule. For the 28-day administration, the Control and model Control groups were intragastrically administered with 2.0 mL of 0.9% sodium chloride daily, the Estradiol group with 2.0 mL of Estradiol suspension (0.2mg/kg/d), and the low dose Kuntai capsule group and the high dose Kuntai capsule group with 2.0 mL of Kuntai capsule suspension (0.6g/kg/d, 1.8g/kg/d, respectively). Sex hormone levels, estrous cycle, and ovarian coefficient of the five groups were compared, histological sections analyzed follicle counts, and the protein expressions of growth differentiation factor 9, light chain 3 A-II, and Beclin 1 in the ovarian tissue were detected by Western blotting. Results: After the 28-day administration, the serum Estradiol and Follicle-Stimulating Hormone levels of the group treated with low dose of Kuntai capsule were not significantly different from the Control group, the serum anti-Müllerian Hormone level of the group treated with high dose of Kuntai capsule was significantly higher than the Estradiol group. The estrous cycle of the group treated with low dose of Kuntai capsule was significantly lower than the model Control group. Regarding ovarian coefficient, resting and growing follicles, growth differentiation factor 9, light chain 3 A-II, and Beclin 1 expression, both Kuntai capsule groups outperformed the model Control group with the statistical difference (P<0.05). Conclusion: Kuntai capsule can improve the estrous cycle and ovarian coefficient of rats with premature ovarian insufficiency, maintain the number of resting and growing follicles, and up-regulate the protein expression of growth differentiation factor 9, light chain 3 A-II, and Beclin 1 of rats' ovaries.

The flavonoid, kaempferol-3-O-apiofuranosyl-7-O-rhamnopyranosyl, as a potential therapeutic agent for breast cancer with a promoting effect on ovarian function.

It is widely known that breast cancer cells eventually develop resistance to hormonal drugs and chemotherapies, which often compromise fertility. This study aimed to investigate the effect of the flavonoid, kaempferol-3-O-apiofuranosyl-7-O-rhamnopyranosyl (KARP), on 1) the viability of MCF-7 breast cancer cells and 2) ovarian function in rats. A dose-dependent decrease in MCF-7 cell survival was observed, and the IC50 value was found to be 48 µg/ml. Cells in the control group or those exposed to increasing concentrations of KARP experienced a similar generation of reactive oxygen species and induction of apoptosis. For the rats, estradiol levels correlated negatively to KARP dosages, although a recovery was obtained at administration of 30 mg/kg per day. Noteworthily, when compared against the control, this dosage led to significant increases in mRNA levels for CYP19, CYP17a, CCND2, GDF9, and INSL3 among the treatment groups, and ER1 and ER2 mRNA levels decreased in a dose-dependent manner. KARP shows great promise as an ideal therapy for breast cancer patients since it induced apoptosis and autophagy in cancerous cells without harming fertility in our animal model. Future investigations on humans are necessary to substantiate these findings and determine its efficacy as a general line of treatment.

Modulatory Effects of Single and Complex Vitamins on the In Vitro Growth of Murine Ovarian Follicles.

Background: Vitamin is a well-known co-factor for many metabolic processes and its roles in fertility and follicular growth have been studied. Vitamin supplementation is frequently achieved by daily ingestion in the form of a complex capsule. However, the role of single and complex vitamins in in vitro maturation of murine follicles is not fully elucidated. Methods: In this study, we evaluated the effects of two forms of vitamins. Pure L-ascorbic acid, and multi-vitamin (vitamin C + vitamin B complex) was treated at two different concentrations (50 and 100 µg/ml), to pre-puberty murine follicles during in vitro maturation. To determine the specific stage of growth that is affected by treatment with vitamins, the vitamins were treated from day 0, 4, 9, and 13. Growth of each follicle was assessed by measuring diameters of whole expanded area and of the granulosa cells. Expression of follicular and oocyte growth-related genes and the effect of vitamin on the viability of follicles was assessed using senescence associated ß-galactosidase staining. Results: Treatment with vitamins promoted the in vitro growth of murine follicles and the upregulated the expression of granulosa cell- and oocyte-specific genes such as BMP15, Fsh receptor, and GDF9. The proliferation of the granulosa cells was enhanced by the treatment of vitamin. Fifty µg/ml concentration vitamin showed greater effects compared to higher concentration. The viability of in vitro grown follicles was also significantly improved in vitamin-treated follicles. The effects of single L-ascorbic acid and complex vitamin were not significantly different to those of day 4 and day 9 follicles. Vitamins promoted murine follicle development in vitro with different effects on specific growth stage. Conclusion: Supplementation of vitamins during in vitro maturation of murine follicles is an efficient strategy for in vitro expansion of follicular cells. These results could be customized to the sophisticated culture of follicles retrieved from aged or cancer-survived female that contain smaller number of follicles with reduced potential to develop into mature follicles.

Growth differentiation factor-9 improves development, mitochondrial activity and meiotic resumption of sheep oocytes after in vitro culture of secondary follicles.

This study analysed the effect of growth differentiation factor-9 (GDF-9) on the in vitro culture of isolated ovine secondary follicles. The follicles were cultured in α-MEM supplemented with BSA, insulin, glutamine, hypoxanthine, transferrin, selenium, ascorbic acid and FSH (α-MEM+ -control medium) or α-MEM+ supplemented with 1, 10, 50 or 100 ng/ml GDF-9. Next, the oocytes were destined to in vitro maturation (IVM). After 12 days of culture, there were no differences regarding the percentage of normal follicles, antrum formation and follicle diameter between the treatments (p > 0.05). The rates of fully grown oocytes (≥110 µm) were higher (p < 0.05) in 100 ng/ml GDF-9 than other treatments, except for 10 ng/ml of GDF-9 (p > 0.05). Treatment containing 100 ng/ml GDF-9 showed higher (p < 0.05) mitochondrial activity than the control group. Moreover, 100 ng/ml GDF-9 showed more oocytes in MI than α-MEM+ , 1 or 50 ng/ml GDF-9 (p < 0.05). In conclusion, 100 ng/ml GDF-9 increased the growth, mitochondrial function and meiotic resumption of oocytes from in vitro grown sheep secondary follicles.

A potential role of knockout serum replacement as a porcine follicular fluid substitute for in vitro maturation: Lipid metabolism approach.

The use of supplements, such as porcine follicular fluid (pFF), fetal bovine serum and human serum albumin are widely used during in vitro maturation (IVM) in different species but these supplements contain undefined components that cause technical difficulties in standardization and influence the efficiency of IVM. Knockout serum replacement (KSR) is a synthetic protein source, without any undefined growth factors or differentiation-promoting factors. Therefore, it is feasible to use KSR as a defined component for avoiding effects of unknown molecules in an IVM system. In this study, the rates of oocyte maturation and blastocyst formation after parthenogenetic activation (PA), somatic cell nuclear transfer (SCNT) and in vitro fertilization (IVF) were significantly higher in the 5% KSR supplemented group than in the unsupplemented control group and more similar to those of the 10% pFF supplemented group. Moreover, the intensity of GDF9, BMP15, ROS, GSH, BODIPY-LD, BODIPY-FA, and BODIPY-ATP staining showed similar values between 5% KSR and 10% pFF, which have significant difference with control group. Most of the gene expression related to lipid metabolism with both supplements exhibited similar patterns. In conclusion, 5% KSR upregulated lipid metabolism and thereby provides an essential energy source to sustain and improve oocyte quality and subsequent embryo development after PA, SCNT, and IVF. These indications support the idea that KSR used as a defined serum supplement for oocyte IVM might be universally used in other species.

Improved developmental competence in embryos treated with lycopene during in vitro culture system.

In vitro embryo development remains suboptimal compared to in vivo development due to the challenge from various stressors associated with in vitro culturing of oocytes. When 0.2 µM lycopene was added to oocyte in vitro maturation and embryo culture media, to assess its antioxidant effects on embryo development, we observed a significant (p < 0.05) increase in cleavage and blastocyst development rates compared to the corresponding controls (84.3 ± 0.6% vs. 73.1 ± 1.9% and 41.0 ± 1.4% vs. 33.4 ± 0.7%, respectively). Lycopene also significantly reduced (p < 0.05) intracellular reactive oxygen species concentrations in oocytes and blastocysts, whereas lipid peroxidation and mitochondrial activity increased compared to control conditions. The number of apoptotic nuclei was significantly reduced in the lycopene-treated compared to the control group (1.7 ± 0.1 vs. 4.7 ± 0.3), and the quantity of cells in the trophectoderm (207.1 ± 1.6 vs. 171.3 ± 1.0, respectively) and inner cell mass (41.9 ± 0.4 vs. 36.7 ± 0.4, respectively) was higher following treatment-although the inner cell mass-to-trophectoderm ratio was unchanged (1:3.3 vs. 1:3.4 for lycopene vs. control, respectively). Lycopene supplementation also significantly (p < 0.05) attenuated expression of IKBKB (Inhibitor of nuclear factor kappa B kinase, subunit beta) and reduced Caspase 9 and Caspase 3 protein abundance, while up-regulating GDF9 (Growth and differentiation factor 9), BMP15 (Bone morphogenetic protein 15), SOD2 (Superoxide dismutase 2), NDUFA2 (NADH dehydrogenase), ACADL (Acyl-CoA dehydrogenase, long chain), and ACSL3 (Acyl-CoA synthetase 3, long-chain membrane 3) transcription compared to control. Therefore, co-culturing with lycopene during oocyte maturation improved bovine embryo developmental potential during in vitro culture by improving embryonic resilience to stress.

Tribulus terrestris Alters the Expression of Growth Differentiation Factor 9 and Bone Morphogenetic Protein 15 in Rabbit Ovaries of Mothers and F1 Female Offspring.

Although previous research has demonstrated the key role of the oocyte-derived factors, bone morphogenetic protein (BMP) 15 and growth differentiation factor (GDF) 9, in follicular development and ovulation, there is a lack of knowledge on the impact of external factors, which females are exposed to during folliculogenesis, on their expression. The present study investigated the effect of the aphrodisiac Tribulus terrestris on the GDF9 and BMP15 expression in the oocytes and cumulus cells at mRNA and protein levels during folliculogenesis in two generations of female rabbits. The experiment was conducted with 28 New Zealand rabbits. Only the diet of the experimental mothers group was supplemented with a dry extract of T. terrestris for the 45 days prior to insemination. The expression of BMP15 and GDF9 genes in the oocytes and cumulus cells of mothers and F1 female offspring was analyzed using real-time polymerase chain reaction (RT-PCR). The localization of the GDF9 and BMP15 proteins in the ovary tissues was determined by immunohistochemical analysis. The BMP15 and GDF9 transcripts were detected in the oocytes and cumulus cells of rabbits from all groups. T. terrestris caused a decrease in the BMP15 mRNA level in the oocytes and an increase in the cumulus cells. The GDF9 mRNA level increased significantly in both oocytes and cumulus cells. The downregulated expression of BMP15 in the treated mothers' oocytes was inherited in the F1 female offspring born to treated mothers. BMP15 and GDF9 show a clearly expressed sensitivity to the bioactive compounds of T. terrestris.

[Dan'e fukang soft extract improved the oocyte quality and GDF-9 expressions of endometriosis patients: an experimental study].

OBJECTIVE: To study the mechanism of Dan'e Fukang Soft Extract (DFSE) on improving oocyte and embryo qualities in endometriosis patients undergoing in vitro fertilization-embryo transfer (IVF-ET). METHODS: Totally 70 patients with endometriosis confirmed by laparoscope were randomly assigned to two groups, the treated group and the control group, 35 cases in each group. Patients in the treated group were treated with DFSE + controlled ovarian hyperstimulation (COH), while those in the control group were treated with DFSE placebo + COH. Besides, recruited were another 35 subjects undergoing intracytoplasmic sperm injection-embryo transfer (ICSI-ET) as a normal control group. The content of growth differentiation factor 9 (GDF-9) in the granulocytes of the mature follicular fluid on the oocyte retrieval day was determined by Western blot. The mRNA expression of GDF-9 was detected by RT-PCR. The oocyte retrieval number, the cleavage rate, the fertilization rate,the high-quality embryo rate, and the pregnancy rate were compared. RESULTS: The mRNA expression of GDF-9 in the granulocytes was significantly higher in the treated group than in the control group, showing statistical difference (P < 0.05), but with no statistical difference when compared with that of the normal control group. There was no statistical difference in the cleavage rate between the two groups (P > 0.05). The fertilization rate and the high-quality embryo rate were higher in the treated group than in the control group, showing statistical difference (P < 0.05), but with no statistical difference when compared with that of the normal control group. CONCLUSIONS: DFSE could improve the oocyte and embryo qualities of endometriosis patients undergoing IVF-ET. Its mechanism might be associated with regulating the GDF-9 mRNA level of granulocytes.

Chemoprotective effect of ascorbic acid, α-tocopherol, and selenium on cyclophosphamide-induced toxicity in the rat ovarium.

OBJECTIVE: The aim of the study was to evaluate the protective efficacy of ascorbic acid, α-tocopherol, and selenium by measuring the glutathione (GSH) levels and proliferating cell nuclear antigen (PCNA) and growth differentiation factor-9 (GDF-9) expression in the ovarian tissues of rats treated with cyclophosphamide (CP) therapy. METHODS: Female Wistar rats were divided into 5 groups of 6 rats each: (I) control, (II) only CP, (III) CP + ascorbic acid, (IV) CP + α-tocopherol, and (V) CP + selenium. Immunohistochemical stainings and GSH protocol were then applied. RESULTS: Following CP administration, the rats exhibited significantly lower GDF-9 expression in oocytes and PCNA expression in granulosa cells of follicles in all stages of development (P < 0.05). In CP + antioxidant groups (Groups III, IV, V), GDF-9 immunoreaction in oocytes and PCNA immunoreaction in granulosa cells of the developing follicles were found to show an increase towards the levels observed in the control group (P < 0.05). CONCLUSIONS: CP was found to cause remarkable degenerative effects in normal ovarian tissue, and we believe that this damage can be reduced and ovarian tissue can be spared from the toxic effects of CP by using antioxidants such as ascorbic acid, α-tocopherol, and selenium.

Impact of gonadotropin supplementation on the expression of germ cell marker genes (MATER, ZAR1, GDF9, and BMP15) during in vitro maturation of buffalo (Bubalus bubalis) oocyte.

The present study was designed to investigate whether gonadotropins [follicle-stimulating hormone (FSH) and luteinizing hormone (LH)] and buffalo follicular fluid (bFF) supplementation in maturation medium influences the transcript abundance of germ cell marker genes [maternal antigen that embryos require (MATER), Zygote arrest 1 (ZAR1), growth differentiation factor 9 (GDF9), and bone morphogenetic protein 15 (BMP15)] mRNA in buffalo (Bubalus bubalis) oocytes. Buffalo ovaries were collected from local abattoir, oocytes were aspirated from antral follicles (5-8 mm) and matured in vitro using two different maturation regimens, viz, group A: gonadotropin (FSH and LH) and group B: non-gonadotropin-supplemented maturation medium containing 20% buffalo follicular fluid (bFF). mRNA was isolated from immature (330) and in vitro matured oocytes from both the groups (A, 320; B, 340), and reverse transcribed using Moloney murine leukemia virus reverse transcriptase. Expression levels of MATER, ZAR1, GDF9, and BMP15 mRNA transcripts were analyzed in oocytes of both maturation groups as well as immature oocytes using real-time PCR. QPCR results showed that GDF9 and BMP15 transcripts were significantly (p<0.05) influenced with gonadotropins and bFF supplementation during in vitro maturation of buffalo oocyte; however, MATER and ZAR1 transcripts were not influenced with gonadotropins and bFF supplementation in vitro. These results indicated that the expression levels of MATER, ZAR1, GDF9, and BMP15 mRNA were varied differentially during in vitro maturation of buffalo oocyte and were found to be gonadotropins (FSH and LH) or bFF dependent for GDF9 and BMP15.

Effects of growth differentiation factor-9 and FSH on in vitro development, viability and mRNA expression in bovine preantral follicles.

The present study investigated the role of growth differentiation factor (GDF)-9 and FSH, alone or in combination, on the growth, viability and mRNA expression of FSH receptor, proliferating cell nuclear antigen (PCNA) and proteoglycan-related factors (i.e., hyaluronan synthase (HAS) 1, HAS2, versican, perlecan) in bovine secondary follicles before and after in vitro culture. After 12 days culture, sequential FSH (100 ng mL⁻¹) from Days 0 to 6 and 500 ng mL⁻¹ from Days 7 to 12) increased follicular diameter and resulted in increased antrum formation (P<0.05). Alone, 200 ng mL⁻¹ GDF-9 significantly reduced HAS1 mRNA levels, but increased versican and perlecan mRNA levels in whole follicles, which included the oocyte, theca and granulosa cells. Together, FSH and GDF-9 increased HAS2 and versican (VCAN) mRNA levels, but decreased PCNA mRNA expression, compared with levels in follicles cultured in α-minimum essential medium supplemented with 3.0 mg mL⁻¹ bovine serum albumin, 10 µg mL⁻¹ insulin, 5.5 µg mL⁻¹ transferrin, 5 ng mL⁻¹ selenium, 2 mM glutamine, 2mM hypoxanthine and 50 µg mL⁻¹ ascorbic acid (α-MEM⁺). Comparisons of uncultured (0.2 mm) and α-MEM⁺ cultured follicles revealed that HAS1 mRNA expression was higher, whereas VCAN expression was lower, in cultured follicles (P<0.05). Expression of HAS1, VCAN and perlecan (HSPG2) was higher in cultured than in vivo-grown (0.3 mm) follicles. In conclusion, FSH and/or GDF-9 promote follicular growth and antrum formation. Moreover, GDF-9 stimulates expression of versican and perlecan and interacts positively with FSH to increase HAS2 expression.

Effect of soothing liver therapy on oocyte quality and growth differentiation factor-9 in patients undergoing in vitro fertilization and embryo transfer.

OBJECTIVE: To investigate the effect of Soothing liver therapy on infertile women undergoing in vitro fertilization and embryo transfer (IVF-ET) and to explore its mechanism. METHODS: Fifty-eight women with tubal infertility were randomized into two groups: 30 in an experimental group treated with Xiaoyao powder (Shugan) plus gonadotropin-releasing hormone analog (GnRHa)/follicle-stimulating hormone (FSH)/ human chorionic gonadotropin (hCG) and 28 in the control group who were treated with GnRHa/FSH/ hCG only. The total gonadotropin (Gn) doses required, endometrial thickness, oocyte numbers, high quality embryo production rate and pregnancy rate of the two groups were compared. The concentration of growth differentiation factor-9 (GDF-9) in follicular fluid was detected by western blotting and the expression of GDF-9 mRNA in granulosa cells was measured using reverse transcription-polymerase chain reaction amplification. RESULTS: In the experimental group, the Gn dose was significantly lower than that in the control group; the endometrial thickness, high quality embryo production and pregnancy rates were significantly higher and the expression of GDF-9 mRNA was also significantly higher than in the control group (all P < 0.05). CONCLUSION: Shugan treatment can improve the pregnancy rate of women with tubal infertility; its mechanism is possibly related to the increased expression of GDF-9 in granulosa cells.

Dan'e fukang soft extract improved the oocyte quality and GDF-9 expressions of endometriosis patients: an experimental study / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study the mechanism of Dan'e Fukang Soft Extract (DFSE) on improving oocyte and embryo qualities in endometriosis patients undergoing in vitro fertilization-embryo transfer (IVF-ET).</p><p><b>METHODS</b>Totally 70 patients with endometriosis confirmed by laparoscope were randomly assigned to two groups, the treated group and the control group, 35 cases in each group. Patients in the treated group were treated with DFSE + controlled ovarian hyperstimulation (COH), while those in the control group were treated with DFSE placebo + COH. Besides, recruited were another 35 subjects undergoing intracytoplasmic sperm injection-embryo transfer (ICSI-ET) as a normal control group. The content of growth differentiation factor 9 (GDF-9) in the granulocytes of the mature follicular fluid on the oocyte retrieval day was determined by Western blot. The mRNA expression of GDF-9 was detected by RT-PCR. The oocyte retrieval number, the cleavage rate, the fertilization rate,the high-quality embryo rate, and the pregnancy rate were compared.</p><p><b>RESULTS</b>The mRNA expression of GDF-9 in the granulocytes was significantly higher in the treated group than in the control group, showing statistical difference (P < 0.05), but with no statistical difference when compared with that of the normal control group. There was no statistical difference in the cleavage rate between the two groups (P > 0.05). The fertilization rate and the high-quality embryo rate were higher in the treated group than in the control group, showing statistical difference (P < 0.05), but with no statistical difference when compared with that of the normal control group.</p><p><b>CONCLUSIONS</b>DFSE could improve the oocyte and embryo qualities of endometriosis patients undergoing IVF-ET. Its mechanism might be associated with regulating the GDF-9 mRNA level of granulocytes.</p>

[Effect of bushen tiaojing recipe on growth differentiation factor-9 in tubal infertility patients undergoing in vitro fertilization-embryo transfer].

OBJECTIVE: To study the effect and mechanism of Bushen Tiaojing Recipe on improving oocyte and embryo qualities in patients undergoing in vitro fertilization-embryo transfer (IVF-ET) at the super-ovulatory cycle. METHODS: Fifty-eight tubal infertility patients undergoing IVF-ET were randomly assigned to two groups. Thirty patients in the treatment group were treated with Bushen Tiaojing Recipe and GnRHa/FSH/hCG, and twenty-eight patients in the control group were treated with GnRHa/FSH/hCG. Contents of GDF-9 in the mature follicular fluid were detected by Western blot. The expressions of GDF-9 in granulose cells were detected by Real-time PCR. The dose of gonadotropin (Gn), the number of oocytes obtained, the fertilization rate, the oocyte cleavage rate, the high quality embryo rate, and the pregnancy rate were compared. RESULTS: The contents of GDF-9 in the follicular fluid and its expression in granulosa cells were significantly higher in the treatment group than in the control group (P<0.05). The number of oocytes obtained, the fertilization rate, the high quality embryo rate, and the pregnancy rate were significantly higher in the treatment group than in the control group. There was no significant difference in the dose of Gn or the oocyte cleavage rate. CONCLUSIONS: Bushen Tiaojing Recipe could improve the pregnancy rate of IVF-ET. Its mechanism might be possibly through regulating the GDF-9 contents in the follicular fluid and granulosa cells.

Temporal effects of exogenous oocyte-secreted factors on bovine oocyte developmental competence during IVM.

We investigated whether paracrine signalling between the bovine oocyte and cumulus cells is altered during the course of in vitro maturation (IVM). Bovine COCs were cocultured with denuded oocytes or treated with specific oocyte-secreted factors, namely recombinant bone morphogenetic protein (BMP)-15 or growth differentiation factor (GDF)-9, beginning from 0 or 9h IVM. To generate a 9-h denuded oocyte (DO) group, COCs were cultured intact for the first 9h of IVM and then denuded. Coculturing intact COCs with DOs denuded immediately after collection or following 9h of maturation did not affect cleavage rate, but improved blastocyst yield (P<0.05) on Day 8 (51 and 61%, respectively; P<0.05) and cell number compared with COCs cultured alone (41%). Significantly, we observed higher levels of endogenous GDF-9 and BMP-15 protein in oocytes of COCs matured for 9h compared with no incubation. The addition of 175 ng mL(-1) GDF-9 or 10%v/v BMP-15 from partially purified transfected 293H cell supernatant for 24h IVM significantly enhanced development to the blastocyst stage from 40% (control) to 51 and 47%, respectively (P<0.05). However, treatment of COCs with GDF-9 or BMP-15 between 9 and 24h of IVM did not increase blastocyst yield. These results provide evidence of quantitative and possibly qualitative temporal changes in oocyte paracrine factor production during IVM.