Abstract
The ovulatory LH-surge increases Vegf gene expression in granulosa cells (GCs) undergoing luteinization during ovulation. To understand the factors involved in this increase, we examined the roles of two transcription factors and epigenetic mechanisms in rat GCs. GCs were obtained from rats treated with eCG before, 4 h, 8 h, 12 h and 24 h after hCG injection. Vegf mRNA levels gradually increased after hCG injection and reached a peak at 12 h. To investigate the mechanism by which Vegf is up-regulated after hCG injection, we focused on C/EBPβ and HIF1α. Their protein expression levels were increased at 12 h. The binding activity of C/EBPβ to the Vegf promoter region increased after hCG injection whereas that of HIF1α did not at this time point. The C/EBPβ binding site had transcriptional activities whereas the HIF1α binding sites did not have transcriptional activities under cAMP stimulation. The levels of H3K9me3 and H3K27me3, which are transcriptional repression markers, decreased in the C/EBPβ binding region after hCG injection. The chromatin structure of this region becomes looser after hCG injection. These results show that C/EBPβ regulates Vegf gene expression with changes in histone modifications and chromatin structure of the promoter region in GCs undergoing luteinization during ovulation.
Highlights
Angiogenesis is required for the corpus luteum (CL) formation[1,2]
We examined by Western blot analyses whether C/EBPβ and HIF1α protein expression levels were increased in rat granulosa cells (GCs) after Human chorionic gonadotropin (hCG) injection
The present study demonstrated that C/EBPβ is an important transcription factor regulating Vegf expression in rat GCs undergoing luteinization during ovulation
Summary
Angiogenesis is required for the corpus luteum (CL) formation[1,2]. After the surge of luteinizing hormone (LH), endothelial cells in the theca cell layer proliferate and start to invade the granulosa cell layer during ovulation. In C/EBPα and β knockout mice, the expressions of angiogenesis-related genes are suppressed and vascular development in CL is defective[12] These findings led us to hypothesize that Vegf expression is regulated by C/EBPβ in GCs during ovulation. Gapdh reported that changes of histone modifications and chromatin remodeling are involved in the regulation of three luteinization-related genes: steroidogenic acute regulatory protein (StAR), aromatase (Cyp19a1) and cytochrome P450 cholesterol side-chain cleavage enzyme (Cyp11a1). These findings raised the possibility that Vegf expression is regulated by transcription factors and by an epigenetic mechanism. We showed that C/EBPβ, but not HIF1α, regulates Vegf expression in rat GCs undergoing luteinization
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