The hypoxic microenvironment that occurs in fast-growing tissue such as the corpus luteum (CL) is a major contributor to its ability to survive via the induction of an intricate vascular network. Cellular responses to hypoxia are mediated by hypoxia-inducible factor-1 (HIF-1), an oxygen-regulated transcriptional activator. HIF-1, a heterodimer consisting of a constitutively-expressed β subunit and an oxygen-regulated α subunit, binds to the hypoxia responsive element (HRE) present in the promoter regions of responsive genes. This review summarises evidence for the involvement of hypoxia and HIF-1α in CL development and function. Special emphasis is given to hypoxia-induced, luteal cell-specific expression of multiple genes (vascular endothelial growth factor A (VEGFA), fibroblast growth factor 2 (FGF-2), prokineticin receptor 2 (PK-R2), stanniocalcin 1 (STC-1) and endothelin 2 (EDN-2) that participate in the angiogenic process during CL formation.

Hypoxia-inducible factor 1 alpha (HIF1A) and endothelin 2 (EDN2) are transiently expressed during the same time window in the developing corpus luteum (CL). In this study, we sought to investigate the involvement of LH/cAMP, reactive oxygen species (ROS), and a hypoxia-mimetic compound (CoCl 2 ) on HIF1A expression and how it affected EDN2 levels, using transformed human granulosa cells (thGCs) and primary bovine granulosa cells (GCs). CoCl 2 elevated HIF1A protein levels in thGCs in a dose-dependent manner. Forskolin alone had no significant effect; however, forskolin and CoCl 2 together further induced HIF1A protein and EDN2 mRNA expression in thGCs. Similarly, in primary GCs, LH with CoCl 2 synergistically augmented HIF1A protein levels, which resulted in higher expression of EDN2 and another well-known hypoxia-inducible gene, VEGF (VEGFA). Importantly, LH alone elevated HIF1A mRNA but not its protein. The successful knockdown of HIF1A in thGCs using siRNA abolished hypoxia-induced EDN2 and also the additive effect of forskolin and CoCl 2 . We then examined the roles of ROS in thGCs: hydrogen peroxide (20 and 50 mM) elevated HIF1A protein as well as the expression of EDN2, implying that induction of HIF1A protein levels is sufficient to stimulate the expression of EDN2 (and VEGF) in normoxia. A broad-range ROS scavenger, butylated hydroxyanisole, inhibited CoCl 2 -induced HIF1A protein with a concomitant reduction in the mRNA expression of EDN2 and VEGF in thGCs. The results obtained in this study suggest that HIF1A, induced by various stimuli, is an essential mediator of EDN2 mRNA expression. The results may also explain the rise in the levels of HIF1A-dependent genes (EDN2 and VEGF) in the developing CL.

Endothelin-2 (EDN2), expressed at a narrow window during the periovulatory period, critically affects ovulation and corpus luteum (CL) formation. LH (acting mainly via cAMP) and hypoxia are implicated in CL formation; therefore, we aimed to elucidate how these signals regulate using human primary (hGLCs) and immortalized (SVOG) granulosa-lutein cells. The hypoxiamiR, microRNA-210 (miR-210) was identified as a new essential player in expression. Hypoxia (either mimetic compound-CoCl, or low O) elevated hypoxia-inducible factor 1A (HIF1A), miR-210 and Hypoxia-induced miR-210 was suppressed in HIF1A-silenced SVOG cells, suggesting that miR-210 is HIF1A dependent. Elevated miR-210 levels in hypoxia or by miR-210 overexpression, increased Conversely, miR-210 inhibition reduced levels, even in the presence of CoCl, indicating the importance of miR-210 in the hypoxic induction of A molecule that destabilizes HIF1A protein, glycerol-3-phosphate dehydrogenase 1-like gene-, was established as a miR-210 target in both cell types. It was decreased by miR-210-mimic and was increased by miR-inhibitor. Furthermore, reducing by endogenously elevated miR-210 (in hypoxia), miR-210-mimic or by siRNA resulted in elevated HIF1A protein and levels, implying a vital role for in the hypoxic induction of Under normoxic conditions, forskolin (adenylyl cyclase activator) triggered changes typical of hypoxia. It elevated, and miR-210 while inhibiting Furthermore, HIF1A silencing greatly reduced forskolin's ability to elevate and miR-210. This study highlights the novel regulatory roles of miR-210 and its gene target, GPD1L, in hypoxia and cAMP-induced by human granulosa-lutein cells.

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