Follicular development and differentiation are sequential events which are tightly regulated by endocrine hormones, intraovarian regulators and cell-cell interactions. Balanced cell proliferation and apoptosis play an important role in the selection of dominant follicle. Primordial germ cell migration and homing within the gonadal ridge requires regulation by integrated signals, such as the oocyte-secreted polypeptide growth factors, the growth and differentiation factor 9, the bone morphogenetic proteins, stem cell factor (SCF), basic fibroblast growth factor (bFGF), the transcription factor Wilms' tumour 1 (Wt1), and involves the contact of primordial germ cells with extra-cellular matrix proteins and cellular substrates and attraction by the developing gonads. Maturation of cumulus-oocyte complexes and ovulation are directly controlled by luteinizing hormone (LH) and require activation of mitogen-activated protein kinase in granulosa cells. In this review, the key molecules involved in the cell-cell interaction and signal transduction during follicular development, differentiation and ovulation will be summarized, mainly focusing on the signaling factors produced by oocyte and the somatic cells.
Cell Differentiation ; China ; Female ; Granulosa Cells ; Growth Differentiation Factor 9 ; Humans ; Mitogen-Activated Protein Kinases ; Oocytes ; Ovary ; Signal Transduction

OBJECTIVE: In vitro maturation (IVM) of immature oocytes can be useful for some infertile patients. In IVM programs, the rates of embryo formation and pregnancy are low. Therefore, it is essential to recognize the main factors involved in regulating oocyte maturation in vitro. The purpose of this study was to investigate the effects of growth differentiation factor 9 (GDF9) and cumulus cell (CC) supplementation in IVM medium on the rates of embryo formation and viability of human blastocysts.METHODS: A total of 80 germinal vesicle oocytes from stimulated cycles underwent an IVM program. The oocytes were divided into four groups, where group I consisted of IVM media only and served as the control, group II consisted of IVM+CCs, group III consisted of IVM+GDF9 (200 ng/mL), and group IV consisted of IVM+CCs+GDF9 (200 ng/mL). Intracytoplasmic sperm injection was performed on the IVM oocytes, and the cleavage embryos that were generated were vitrified. Following thawing, the embryos were cultured for 3 additional days, and the viability rates of the developed blastocysts were determined.RESULTS: The maturation rate of the oocytes did not differ significantly across the four groups. The fertilization rate in group II was significantly higher than that in the control group (76.5% vs. 46.2%). Embryo formation was significantly more frequent in all experimental groups than in the control group, while blastocyst formation did not show significant differences in the three experimental groups compared to the control. The mean viability rates in groups II, III, and IV were 58.16%, 55.91%, and 55.95%, respectively, versus 37.78% in the control group (p<0.05).CONCLUSION: Supplementation of IVM culture media with GDF9 and CCs enhanced the fertilization, embryo formation, and viability rates of blastocysts generated from vitrified cleavage embryos.
Blastocyst ; Culture Media ; Cumulus Cells ; Embryonic Structures ; Fertilization ; Growth Differentiation Factor 9 ; Humans ; In Vitro Techniques ; Oocytes ; Pregnancy ; Sperm Injections, Intracytoplasmic

OBJECTIVE: This study was performed to identify whether growth and differentiation factor-9 (GDF-9) and transforming growth factor-beta1 (TGF-beta1) expressions would be lower in the follicular fluid (FF) of those over age 35 who underwent IVF than under age 35. METHODS: A total of 24 IVF cycles (20 patients) were included in this study. All of patients were stimulated for IVF by the GnRH short protocol and divided into two groups for analysis, according to their age: <35 group (14 cycles, 11 patients) vs. > or =35 group (10 cycles, 9 patients). The expression levels of GDF-9 and TGF-beta1 were determined by western blotting and quantitative enzyme-linked immunosorbent assay. RESULTS: The numbers of retrieved oocytes and metaphase II oocytes were significantly lower in the > or =35 group. Lower expression of GDF-9 and TGF-beta1 by western blotting in the > or =35 group were observed as well. The mean GDF-9 and TGF-beta1 levels by enzyme-linked immunosorbent assay were lower in the > or =35 group. The values were 6,850.5+/-928.4 ng/L vs. 3,333.3+/-1,089.2 ng/L of GDF-9 (p<0.05) and 3,844.1+/-571.1 ng/L vs. 2,187.7+/-754.0 ng/L of TGF-beta1 (p<0.05). A negative correlation between GDF-9 and age was observed (r=-0.546, p=0.006). CONCLUSION: GDF-9 and TGF-beta1 production from stimulated ovaries during IVF appears to decrease with age.
Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Female ; Fertilization ; Follicular Fluid ; Gonadotropin-Releasing Hormone ; Growth Differentiation Factor 9 ; Humans ; Metaphase ; Oocytes ; Ovary ; Transforming Growth Factor beta1

OBJECTIVETo assess the effect of repeated gonadotropic stimulations on the developmental potential and growth differentiation factor-9 (GDF-9) expression of mouse oocytes.

METHODSFemale Kunming mice were treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG) for 3 times, and the control mice were treated with normal saline. The two groups of mice were both stimulated subsequently to obtain the mature oocytes. Immunocytochemical staining was employed to evaluate GDF-9 expression in the oocytes. The oocytes were then inseminated and cultured till the formation of blastocysts to compare the cleavage rate and blastocyst formation rate between the groups.

RESULTSA total of 253 mature oocytes were obtained in the repeated stimulation group, with a mean of 11.5 oocytes from each mouse; 521 mature oocytes were obtained in the control group with a significantly greater mean number of 32.6 from each mouse (P<0.05). The average optical density and integrated optical density for GDF-9 expression were significantly lower in the oocytes in repeated stimulation group than in the control group (P<0.05 and 0.01, respectively). After insemination, the cleavage rate were comparable between repeated stimulation group and the control group (85.6% vs 88.8%), but the blastocyst formation rate was significantly lower in repeated stimulation group (20.8% vs 35.2%, P<0.01).

CONCLUSIONRepeated gonadal stimulation decreases the developmental potential of mouse oocytes possibly due to reduced GDF-9 expression.

Animals ; Cells, Cultured ; Female ; Gonadotropins ; pharmacology ; Growth Differentiation Factor 9 ; metabolism ; Mice ; Oocytes ; cytology ; growth & development ; metabolism ; Ovulation Induction ; adverse effects ; methods

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.
Adult ; Amenorrhea ; Bone Morphogenetic Protein 15 ; Chromatography, High Pressure Liquid ; Female ; Gonadotropins ; Growth Differentiation Factor 9 ; Humans ; Inhibins ; Mass Screening ; Primary Ovarian Insufficiency* ; Receptors, FSH ; Receptors, LH

OBJECTIVETo study the roles of matrix metalloproteinases-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), vascular endothelial growth factor (VEGF) and transforming growth factor β-1 (TGFβ-1) in differentiation, invasiveness and metastatic potential of papillary carcinoma and follicular carcinoma of thyroid.

METHODSEighty-five cases of papillary thyroid carcinoma and 59 cases of follicular thyroid carcinoma were enrolled into the study. Immunohistochemistry using EnVision method was carried out for assessment of the expression of MMP-9, TIMP-1, VEGF and TGFβ-1 in the tumor tissue.

RESULTSMMP-9, TIMP-1, VEGF and TGFβ-1 were expressed in the cytoplasm of tumor cells. The positivity rates of MMP-9, TIMP-1, VEGF and TGFβ-1 in papillary thyroid carcinoma (83.5%, 81.2%, 90.6% and 75.3%, respectively) were similar to or lower than those in follicular thyroid carcinoma (93.2%, 86.4%, 89.9% and 78.0%, respectively). The expression rates in papillary thyroid carcinoma with lymph node metastasis were also higher than those in tumors without lymph node metastasis. The expression rates of MMP-9, VEGF and TGFβ-1 in poorly-differentiated follicular thyroid carcinoma were higher than those in well-differentiated follicular thyroid carcinoma. The expression of TIMP-1 however showed a negative correlation with the tumor cell differentiation. In general, the expression of VEGF and MMP-9 was higher than that of TIMP-1 and TGFβ-1 in papillary thyroid carcinoma and follicular thyroid carcinoma.

CONCLUSIONSImmunohistochemical detection of MMP-9, TIMP-1, VEGF and TGFβ-1 expression may carry clinical significance in evaluating the degree of differentiation, invasiveness, metastatic potential and prognosis of papillary thyroid carcinoma and follicular thyroid carcinoma.

Adenocarcinoma, Follicular ; metabolism ; pathology ; Carcinoma, Papillary ; metabolism ; pathology ; Cell Differentiation ; Humans ; Lymphatic Metastasis ; Matrix Metalloproteinase 9 ; metabolism ; Neoplasm Invasiveness ; Thyroid Neoplasms ; metabolism ; pathology ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism ; Transforming Growth Factor beta1 ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism

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.
Animals ; Anti-Mullerian Hormone ; Female ; Granulosa Cells ; Growth Differentiation Factor 9 ; Humans ; Infant, Newborn ; Intercellular Signaling Peptides and Proteins ; Mental Competency ; Mice ; Oocytes ; Ovary ; Real-Time Polymerase Chain Reaction ; Receptors, FSH ; RNA, Messenger*

OBJECTIVETo 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).

METHODSTotally 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.

RESULTSThe 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.

CONCLUSIONSDFSE 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.

Adult ; Drugs, Chinese Herbal ; therapeutic use ; Embryo Transfer ; Endometriosis ; metabolism ; therapy ; Female ; Fertilization in Vitro ; Growth Differentiation Factor 9 ; metabolism ; Humans ; Infertility, Female ; metabolism ; therapy ; Oocyte Retrieval ; Oocytes ; cytology ; Phytotherapy ; Pregnancy ; Sperm Injections, Intracytoplasmic ; Young Adult

OBJECTIVETo 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.

METHODSFifty-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.

RESULTSThe 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.

CONCLUSIONSBushen 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.

Adult ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Embryo Transfer ; Fallopian Tube Diseases ; complications ; Female ; Fertilization in Vitro ; Granulosa Cells ; drug effects ; metabolism ; Growth Differentiation Factor 9 ; metabolism ; Humans ; Infertility, Female ; etiology ; metabolism ; therapy ; Phytotherapy ; Young Adult

OBJECTIVETo explore the relation between oocyte maturation and growth differentiation factor-9 (GDF-9) gene expression.

METHODSOvariectomy was performed in 50 Kunming female mice of 10 days old, and the preantral follicles were isolated from the ovaries and cultured in medium drops for 12 days. Oocytes and somatic cells were mechanically isolated. The oocytes cultured in vitro for 2, 4, 6, 8, 10, and 12 days constituted the in vitro cultured group and the oocytes obtained from female mice of 12, 14, 16, 18, 20, and 22 days old served as the in vivo group. Semi-quantitative RT-PCR and agar gel electrophoresis were performed to quantify GDF-9 gene expression in each oocyte.

RESULTSFollicle survival, antrum formation and maturation rate was 89.5%, 51.8% and 56.6% in the in vitro cultured follicles, respectively. GDF-9 gene expression on days 2, 4, 6, 8, 10, and 12 in in vitro cultured oocytes was 0.83-/+0.08, 0.52-/+0.09, 0.45-/+0.13, 0.49-/+0.09, 0.49-/+0.09, and 0.68-/+0.08, respectively; GDF-9 gene expression in in vivo grown oocytes of 12, 14, 16, 18, 20, and 22 days were 0.64-/+0.35, 0.48-/+0.10, 0.52-/+0.10, 0.66-/+0.08, 0.72-/+0.09, and 0.91-/+0.11, respectively. Between days 8 and 12, GDF-9 gene expression in in vitro cultured oocyte was significantly lower than that in in vivo grown oocytes (P<0.05).

CONCLUSIONMII oocytes can be obtained from in vitro culture of the preantral follicles. GDF-9 gene expression in the oocytes varies with their growth stages. Between days 8 and 12 of in vitro culture, GDF-9 gene expression in the cultured oocytes is different from that in in vivo grown oocytes.

Animals ; Animals, Newborn ; Bone Morphogenetic Protein 15 ; Cell Survival ; Cells, Cultured ; Electrophoresis, Agar Gel ; Female ; Gene Expression ; Growth Differentiation Factor 9 ; Intercellular Signaling Peptides and Proteins ; genetics ; Mice ; Oocytes ; cytology ; growth & development ; metabolism ; Ovarian Follicle ; cytology ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors