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.

Growth differentiation factor 9 and cumulus cell supplementation in in vitro maturation culture media enhances the viability of human blastocysts / 대한생식의학회지

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.

Advanced studies on ovary physiology in China in the past 30 years / 生理学报

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.

A spatial model showing differences between juxtacrine and paracrine mutual oocyte-granulosa cells interactions.

The bidirectional communication between oocytes and granulosa cells are mediated by several factors via a local feedback loop(s). The current model was carried out to study the spatial mutual interaction of porcine denuded oocytes and granulosa cells either in direct contact (juxtacrine) or paracrine co-culture using transwell system. Transwell 0.4 µm polyester membrane inserts were used to permit oocytes-granulosa cells paracrine communication with a distance of 2 mm between them in co-culture. Oocytes were cultured with granulosa cells in a defined basic maturation medium for 44 h. In results, oocyte secreted factors (OSFs; GDF9 and BMP15) temporal expression showed progressive decrement by the end of culture in case of direct contact with granulosa cells while it was increased progressively in the paracrine co-culture groups. However, oocytes that were cultured in direct contact showed a significant increase in blastocyst development after parthenogenetic activation than the paracrine co-cultured ones (20% vs. 11.5%, respectively). By the end of culture, granulosa cell count in direct contact showed a significant decrease than the indirect co-culture group (1.2 × 105 cell/mL vs. 2.1 × 105 cell/mL, respectively). Steroids (P4 and E2) and steriodogenesis enzymes mRNA levels showed significant temporal alterations either after 22 h and 44 h of IVM in both juxtacrine and paracrine co-culture systems (P ≤ 0.05). CX43 was much more highly expressed in the granulosa of the direct contact group than the indirect co-culture group. These results indicate the difference in mutual communication between oocytes and granulosa cells that were cocultured either in direct contact (juxtacrine) or with a short distance (paracrine) and propose a new paradigm to study different ovarian follicular cells interaction.

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.

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>

Lower growth factor expression in follicular fluid undergone in-vitro fertilization / 대한생식의학회지

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: 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.

Effect of bushen tiaojing recipe on growth differentiation factor-9 in tubal infertility patients undergoing in vitro fertilization-embryo transfer / 中国中西医结合杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>

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.

Repeated gonadotropin stimulations lower the developmental potential of mouse oocytes / 南方医科大学学报

<p><b>OBJECTIVE</b>To assess the effect of repeated gonadotropic stimulations on the developmental potential and growth differentiation factor-9 (GDF-9) expression of mouse oocytes.</p><p><b>METHODS</b>Female 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.</p><p><b>RESULTS</b>A 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).</p><p><b>CONCLUSION</b>Repeated gonadal stimulation decreases the developmental potential of mouse oocytes possibly due to reduced GDF-9 expression.</p>

Women with poor response to ovarian stimulation have increased follicular bone morphogenetic protein-15 levels / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To evaluate the levels of bone morphogenetic protein-15 (BMP-15) in human follicular fluid (FF) and its association with response to ovarian stimulation.</p><p><b>METHODS</b>Western blotting was performed to determine the levels of BMP-15 in FF obtained from follicle aspirates in 70 patients undergoing IVF treatment. According to the response to ovarian stimulation the patients were divided into poor responder group and normal responder group.</p><p><b>RESULT</b>BMP-15 levels in FF of poor responders were significantly higher than those in normal responders (1.01 +/- 0.34 vs 0.77 +/- 0.24, P<0.01).</p><p><b>CONCLUSION</b>Increased levels of BMP-15 in FF may be associated with poor response to ovarian stimulation.</p>

Growth differentiation factor-9 gene expression in in vitro cultured oocytes in mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the relation between oocyte maturation and growth differentiation factor-9 (GDF-9) gene expression.</p><p><b>METHODS</b>Ovariectomy 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.</p><p><b>RESULTS</b>Follicle 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).</p><p><b>CONCLUSION</b>MII 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.</p>

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.