Reproduction Abstracts

Introduction: Hypoxia and Endothelin-2 (EDN2) peak simultaneously during early corpus luteum (CL) formation, suggesting causal relationship. Indeed, hypoxia inducible factor-1 alpha (HIF1A) is a strong stimulator of EDN2 in granulosa–lutein cells (GLCs). Knockout of EDN2 resulted in un-ruptured follicles that failed to develop into CL, suggesting that EDN2 plays essential roles during ovulation. In agreement, EDN2 was low in GLCs of women with PCOS, characterized by ovulatory dysfunction. Hypoxia also induce miR-210, a prototypic hypoxiamiR. However, whether miR-210 affects EDN2 mRNA is unknown yet. We explored here the molecular interactions between miR-210 and EDN2 in several cell models.

Methods: Immortalized or primary GLCs from normal or PCOS women undergoing IVF were studied. Hadassah Review Board approved the study (HMO-0110-09) and women gave informed consent. Cells were incubated in hypoxic and normoxic conditions, miR-210 levels were manipulated and genes were silenced using siRNA. mRNAs and miR-210 were measured by qPCR. HIF1A protein was determine by Western blot.

Results and Discussion: miR-210 and EDN2 were closely related in vivo and in vitro: hypoxia and miR-210 overexpression both increased EDN2, while miR-210 inhibition reduced EDN2 in immortalized GLC. Also in GLC from PCOS patients, low miR-210 and EDN2 were noted. Furthermore, HIF1A-silenced cells, with decreased EDN2, similarly exhibited lower miR-210. Glycerol-3-phosphate dehydrogenase 1-like (GPD1L) was identified as a gene target of miR-210 lowering its levels either by miR-210 overexpression or siRNA knockdown, increased HIF1A protein and EDN2 levels. Together, these results propose a positive feedback regulatory loop, where miR-210 induced by hypoxia (via HIF1A) lowers GPD1L which further elevate and maintain HIF1A protein and EDN2 levels. This feed forward loop is expected to boost EDN2 in hGLCs exposed to hypoxia during ovulation. On the other hand, reduced miR-210 in PCOS might interrupt the loop, decrease EDN2 and impair ovulation.