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Ketan Shrestha Department of Animal Sciences, The Robert H. Smith Faculty of Agriculture, Food, and Environment, The Hebrew University of Jerusalem, Rehovot, Israel

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Adepeju Esther Onasanya Department of Animal Sciences, The Robert H. Smith Faculty of Agriculture, Food, and Environment, The Hebrew University of Jerusalem, Rehovot, Israel

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Iris Eisenberg The Magda and Richard Hoffman Center for Human Placenta Research, Hadassah Hebrew University Medical Center, Jerusalem, Israel

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Noa Wigoda Department of Animal Sciences, The Robert H. Smith Faculty of Agriculture, Food, and Environment, The Hebrew University of Jerusalem, Rehovot, Israel

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Simcha Yagel The Magda and Richard Hoffman Center for Human Placenta Research, Hadassah Hebrew University Medical Center, Jerusalem, Israel
Department of Obstetrics and Gynecology, Hadassah Hebrew University Medical Center, Jerusalem, Israel

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Ronit Yalu Department of Animal Sciences, The Robert H. Smith Faculty of Agriculture, Food, and Environment, The Hebrew University of Jerusalem, Rehovot, Israel

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Rina Meidan Department of Animal Sciences, The Robert H. Smith Faculty of Agriculture, Food, and Environment, The Hebrew University of Jerusalem, Rehovot, Israel

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Tal Imbar The Magda and Richard Hoffman Center for Human Placenta Research, Hadassah Hebrew University Medical Center, Jerusalem, Israel
Department of Obstetrics and Gynecology, Hadassah Hebrew University Medical Center, Jerusalem, Israel

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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 EDN2 using human primary (hGLCs) and immortalized (SVOG) granulosa-lutein cells. The hypoxiamiR, microRNA-210 (miR-210) was identified as a new essential player in EDN2 expression. Hypoxia (either mimetic compound-CoCl2, or low O2) elevated hypoxia-inducible factor 1A (HIF1A), miR-210 and EDN2. 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 EDN2. Conversely, miR-210 inhibition reduced EDN2 levels, even in the presence of CoCl2, indicating the importance of miR-210 in the hypoxic induction of EDN2. A molecule that destabilizes HIF1A protein, glycerol-3-phosphate dehydrogenase 1-like gene-GPD1L, 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 GPD1L by endogenously elevated miR-210 (in hypoxia), miR-210-mimic or by GPD1L siRNA resulted in elevated HIF1A protein and EDN2 levels, implying a vital role for GPD1L in the hypoxic induction of EDN2. Under normoxic conditions, forskolin (adenylyl cyclase activator) triggered changes typical of hypoxia. It elevated HIF1A, EDN2 and miR-210 while inhibiting GPD1L. Furthermore, HIF1A silencing greatly reduced forskolin’s ability to elevate EDN2 and miR-210. This study highlights the novel regulatory roles of miR-210 and its gene target, GPD1L, in hypoxia and cAMP-induced EDN2 by human granulosa-lutein cells.

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