Growth differentiation factor 9 inhibits vascular endothelial growth factor expression in human granulosa cells

Abstract

In aortic endothelial cells, the TGFβ signaling pathway is involved in the regulation of vascular endothelial growth factor (VEGF), which encodes a potent angiogenic factor crucial for the development of ovarian hyperstimulation syndrome. Growth differentiation factor 9 (GDF9) is a member of the TGFβ family and its effect on VEGF expression in human granulosa cells is unknown. In this study, human granulosa cells were collected from patients during the course of oocyte retrieval for in vitro fertilization and were cultured in vitro. After the first 48 h of culture, cells were treated with GDF9 with or without SB431542 (an ALK5 inhibitor) at various doses. The medium was then collected to determine the concentration of VEGF by ELISA. Cellular RNA was collected and extracted for quantification by real-time quantitative fluorescence PCR. Our study showed that GDF9 suppressed VEGF release from human granulosa cells in a dose-dependent manner and also downregulated VEGF mRNA levels in these cells. Furthermore, SB431542 antagonized the suppression of VEGF mRNA by GDF9 and diminished the inhibitory effect of GDF9 on VEGF release by human granulosa cells. Our results indicated that GDF9 can inhibit VEGF expression in human granulosa cells and ALK5 might mediate this process.

Chinese abstract

在主动脉内皮细胞中, TGFβ信号通路参与调节血管内皮生长因子（VEGF）, 它编码一种对卵巢过度刺激综合征的发生至关重要的强血管生成因子。生长分化因子9 （Growth differentiation factor 9, GDF9）是TGFβ家族的成员, 其对人颗粒细胞中VEGF表达的影响尚不清楚。本研究在卵母细胞提取过程中, 从患者身上采集人颗粒细胞进行体外培养。培养48小时后, 在加或不加SB431542（一种ALK5抑制剂）情况下, 用不同剂量的GDF9处理细胞。收集培养基, 用ELISA法测定VEGF浓度。收集细胞RNA并通过实时定量荧光PCR提取用于定量。我们的研究表明, GDF9以剂量依赖的方式抑制了人颗粒细胞中VEGF的释放, 并下调了这些细胞中的VEGF mRNA水平。此外, SB431542拮抗了GDF9对VEGF mRNA的抑制, 减弱了GDF9对人颗粒细胞释放VEGF的抑制作用。我们的结果表明, GDF9可以抑制人颗粒细胞中VEGF的表达, 而ALK5可能介导了这一过程。

Keywords:

• Growth differentiation factor 9
• vascular endothelial growth factor
• human granulosa cells
• ovarian hyperstimulation syndrome

Acknowledgements

We thank Luyan Guo, Shan Xiao, and Shuhua Zhu for assistance in our experiments, and colleagues from our embryonic laboratory for help with sample collection.

Disclosure statement

The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Additional information

Funding

This work was funding by the National Natural Science Foundation of China [81370765], National Natural Science Foundation for Young Scientists of China [81601239], Guangdong Provincial Key Laboratory of Reproductive Medicine, Science and Technology Planning Project of Guangdong Province [2012A061400003], and the Pilot Construction of Reproductive Clinical Research and Transformation Center of Guangzhou [155700011/201508020006].