Effects Of Growth Differentiation Factor Research Articles

Effects of Growth Differentiation Factor 9 and Bone Morphogenetic Protein 15 on the in vitro Maturation of Porcine Oocytes

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are members of the transforming growth factor-β (TGF-β) family, and their roles in oocyte maturation and cumulus expansion are well known in the mouse and human, but not in the pig. We investigated GDF9 and BMP15 expressions in porcine oocytes during in vitro maturation. A significant increase in the mRNA levels of GDF9 and BMP15 was observed at germinal vesicle breakdown, with expression levels peaking at metaphase I (MI), but decreasing at metaphase II (MII). GDF9 and BMP15 protein localized to the oocyte cytoplasm. While treatment with GDF9 and BMP15 increased the expression of genes involved in both oocyte maturation (c-mos, cyclinb1 and cdc2) and cumulus expansion (has2, ptgs2, ptx3 and tnfaip6), SB431542 (a TGFβ-GDF9 inhibitor) decreased meiotic maturation at MII. Following parthenogenetic activation, the percentage of blastocysts in SB431542 treatment was lower than in the control (41.3% and 74.4%, respectively). Treatment with GDF9 and BMP15 also increased the mRNA levels of maternal genes such as c-mos [a regulatory subunit of mitogen-activated protein kinase (MAPK)], and cyclinb1 and cdc2 [regulatory subunits of maturation/M-phase-promoting factor (MPF)]; however, SB431542 significantly decreased their mRNA levels. These data were supported by poly (A)-test PCR and protein activity analyses. Our results show that GDF9 and BMP15 participate in cumulus expansion and that they stimulate MPF and MAPK activities in porcine oocytes during in vitro maturation.

Dynamic medium containing growth differentiation factor-9 and FSH maintains survival and promotes in vitro growth of caprine preantral follicles after long-term in vitro culture

The aim of the present study was to evaluate the effects of growth differentiation factor 9 (GDF-9) and FSH on the in vitro development of caprine preantral follicles cultured for 16 days. Ovarian fragments were cultured in αMEM⁺ (α-minimum essential medium, pH 7.2-7.4, 10 μg mL⁻¹ insulin, 5.5 μg mL⁻¹ transferrin, 5.0 ng mL⁻¹ selenium, 2 mM glutamine, 2 mM hypoxanthine and 1.25 mg mL⁻¹ bovine serum albumin) in the absence or presence of 200 ng mL⁻¹ GDF-9 and/or 50 ng mL⁻¹ FSH added during the first (Days 0-8) and/or second (Days 8-16) half of the culture period. Non-cultured and cultured fragments were processed for histological and ultrastructural analyses. After 16 days, all treatments using GDF-9 or FSH showed higher rates of follicular survival compared with αMEM⁺ alone. Compared with non-cultured control, sequential culture media containing GDF-9 and/or FSH significantly increased the percentage of developing follicles and follicle diameter. Moreover, a progressive increase in oocyte diameter was observed only with sequential culture medium containing GDF-9 until Day 8 followed by FSH (GDF-9/FSH) in the second half of the culture period. After 16 days of culture, ultrastructural analysis confirmed the integrity of follicles cultured in the presence of GDF-9/FSH. In conclusion, a dynamic medium containing GDF-9 and FSH (GDF-9/FSH) maintained follicular integrity and promoted activation of primordial follicles and growth during long-term in vitro culture of goat preantral follicles.

Effect of Growth Differentiation Factor 5 on the Proliferation and Tenogenic Differentiation Potential of Human Mesenchymal Stem Cells in vitro

The use of growth differentiation factor 5 (GDF-5) in damaged tendons has been shown to improve tendon repair. It has been hypothesized that further improvements may be achieved when GDF-5 is used to promote cell proliferation and induce tenogenic differentiation in human bone marrow-derived mesenchymal stem cells (hMSCs). However, the optimal conditions required to produce these effects on hMSCs have not been demonstrated in previous studies. A study to determine cell proliferation and tenogenic differentiation in hMSCs exposed to different concentrations of GDF-5 (0, 5, 25, 50, 100 and 500 ng/ml) was thus conducted. No significant changes were observed in the cell proliferation rate in hMSCs treated at different concentrations of GDF-5. GDF-5 appeared to induce tenogenic differentiation at 100 ng/ml, as reflected by (1) a significant increase in total collagen expression, similar to that of the primary native human tenocyte culture; (2) a significant upregulation in candidate tenogenic marker gene expression, i.e. scleraxis, tenascin-C and type-I collagen; (3) the ratio of type-I collagen to type-III collagen expression was elevated to levels similar to that of human tenocyte cultures, and (4) a significant downregulation of the non-tenogenic marker genes runt-related transcription factor 2 and sex determining region Y (SRY)-box 9 at day 7 of GDF-5 induction, further excluding hMSC differentiation into other lineages. In conclusion, GDF-5 does not alter the proliferation rates of hMSCs, but, instead, induces an optimal tenogenic differentiation response at 100 ng/ml.

Effect of GDF‐5 and BMP‐2 on the expression of tendo/ligamentogenesis‐related markers in human PDL‐derived cells

The effect of growth differentiation factor 5 and bone morphogenetic protein 2 on human periodontal ligament-derived cells was investigated with special reference to tendo/ligamentogenesis-related markers. Effects of each factor were analyzed by quantitative PCR for scleraxis and tenomodulin and by western blotting for scleraxis. After exposure to those factors, STRO-1-positive and STRO-1-negative fractions of human periodontal ligament tissues were isolated with an immunomagnetic cell sorting system, and the expression of scleraxis in each fraction was analyzed by western blotting. Non-separated crude cells were used as a control. Growth differentiation factor 5 and bone morphogenetic protein 2 did not increase alkaline phosphatase activity in crude periodontal ligament-derived cells. Growth differentiation factor 5, but not bone morphogenetic protein 2, increased the expression of scleraxis in crude, STRO-1-positive and STRO-1-negative periodontal ligament-derived cells. The expression of scleraxis in STRO-1-positive periodontal ligament-derived cells was significantly less compared to that in crude P2 and STRO-1-negative periodontal ligament-derived cells. Growth differentiation factor 5 induced the expression of scleraxis and may enhance tendo/ligamentogenesis in human periodontal ligament-derived cells. The expression of scleraxis was higher in STRO-1-negative fraction, suggesting more differentiated state of the cells.

Effects of growth differentiation factor 9 on cell cycle regulators and ERK42/44 in human granulosa cell proliferation

GDF-9 stimulates granulosa cell proliferation and plays important roles during folliclogenesis. However, its molecular mechanisms are still far from clear, particularly its roles in human granulosa cells around the periovulatory stage. Therefore, we investigated the effects of GDF-9 on cell cycle distribution, regulatory molecules, and signaling pathways involved in human luteinized granulosa (hLG) cells in vitro. Primary cultures of hLG cells obtained from women undergoing IVF and treated with and without recombinant GDF-9 were evaluated with and without a specific inhibitor to activin receptor-like kinase 5 (ALK5; SB-431542), ERK42/44 (PD-098059), or Smad3 (SIS3). Cell proliferation, cell cycle distribution, mRNA expression, and protein expression of relevant cell cycle molecules were determined by [(3)H]thymidine incorporation, flow cytometry, quantitative PCR, and immunoblotting, respectively. GDF-9 stimulated [(3)H]thymidine incorporation, enhanced cell transition from G(0)/G(1) to S and G(2)/M phases (whereas both SB-431542 and PD-098059 attenuated these changes), increased mRNA and protein expression of cyclin D(1) and E, and decreased those of the cyclin-dependent kinase (CDK) inhibitors p15(INK4B) and p16(INK4A). GDF-9 also activated Rb protein (a critical G(1) to S-phase regulator), ERK42/44, and Smad3. PD-098059 blocked Rb protein phorsphorylation and the increase in cyclin D(1) and E but not the decrease in p15(INK4B) and p16(INK4A) induced by GDF-9. In contrast, SIS3 reversed the decrease in p15(INK4B) and p16(INK4A) but not the increase in cyclin D(1) and E induced by GDF-9. GDF-9 stimulates hLG cell proliferation by stimulating cyclin D(1) and E and suppressing p15(INK4B) and p16(INK4A) via both Smad-dependent and Smad-independent pathways.

Expression and putative functions of GDF-15, a member of the TGF-β superfamily, in human glioma and glioblastoma cell lines

Recent studies have demonstrated growth-inhibiting effects of growth differentiation factor-15 (GDF-15) on different cancer cell lines in vitro and on tumor growth in vivo. Here, we present data concerning expression of GDF-15 in glioblastoma. We found low levels of GDF-15 transcripts in primary glioblastoma. Thus, GDF-15 expression might be exploited as a useful indicator for distinguishing primary from other glial derived tumors. In contrast to the documented proapoptotic and anti-tumorigenic activities of GDF-15 in several cancer cell lines, our data suggest that GDF-15 does not decrease proliferation of glioblastoma cell lines, while its effects on invasiveness are not consistent.

Effect of growth differentiation factor 5 on connexin 43 expression during chondrogenic differentiation of mouse bone marrow stemal cells in vitro

目的 探讨生长分化因子-5(GDF-5)在小鼠骨髓基质干细胞(BMSCs)向软骨分化过程中对缝隙连接蛋白43(Cx43)表达的影响.方法 体外培养小鼠BMSCs,贴壁细胞传代,取第3代细胞,加入地塞米松、VitC、胰岛素和GDF-5诱导培养,72 h后免疫细胞化学和阿尔辛蓝染色分别检测软骨细胞特异性Ⅱ型胶原和蛋白多糖的表达;在诱导24、48和72 h后进行如下检测:MTT法测定GDF-5对小鼠BMSCs增殖的影响;RT-PCR、Western blotting和免疫细胞化学法检测GDF-5对Cx43蛋白表达的影响.结果 MTT结果显示不同时间GDF-5对小鼠BMSCs的增殖无影响;RT-PCR、Western blotting和免疫细胞化学结果表明诱导后不同时间均有Cx43 mRNA和蛋白的表达;诱导72 h免疫细胞化学显示有Ⅱ型胶原蛋白的表达,阿尔辛蓝染色阳性,有蛋白多糖基质的分泌.结论 GDF-5可以通过上调缝隙连接蛋白Cx43的表达来促进小鼠BMSCs向软骨方向的分化。