Signaling by hypoxia-inducible factors is critical for ovulation in mice

The steroid hormone progesterone, acting via its nuclear receptor, is a major regulator of the process of ovulation. Female mice lacking progesterone receptor (PGR) exhibit an anovulatory phenotype due to failure in follicular rupture. To identify the PGR-regulated pathways that control ovulation, we analyzed global changes in gene expression in the ovaries of wild-type and Pgr-null mice subjected to gonadotropin-induced superovulation. Our analysis uncovered several genes whose expression was reduced in the Pgr-null ovaries compared with the wild-type ovaries immediately preceding ovulation. Interestingly, these genes included three hypoxia-inducible factors (HIFs): HIF-1 alpha, HIF-2 alpha, and HIF-1 beta. These transcription factors form alphabeta-heterodimers, which regulate the transcription of specific cellular genes, thereby mediating adaptive response of the tissue to low-oxygen levels. We observed that the expression of mRNAs and proteins corresponding to HIF-1 alpha, HIF-2 alpha, and HIF-1 beta was induced in a PGR-dependent manner, specifically in the granulosa cells of the preovulatory follicles. Inhibition of the HIF transcriptional activity by echinomycin, a small-molecule inhibitor that suppresses the binding of HIF alphabeta-heterodimers to target genes, blocked ovulation by preventing the rupture of the preovulatory follicles. Echinomycin specifically inhibited the expression of genes that are known regulators of ovulation, such as a disintegrin and metalloproteinase with thrombospondin-like motifs-1 and endothelin-2. Furthermore, echinomycin reduced the expression of vascular endothelial growth factor A, a key factor controlling vascularization/angiogenesis during ovulation. Collectively, these findings unveiled a novel ovarian role for the HIF transcription factors during the ovulatory period in mice.