Hormonally regulated follicle differentiation and luteinization in the mouse is associated with hypoxia inducible factor activity

Abstract

Hypoxia inducible factors (HIFs) are transcription factors that mediate physiological responses to hypoxia. Hypoxia is established as the major inducer of HIFs, but stimuli such as transition metals and hormones also induce HIF target genes. Whilst the ovarian granulosa cell layer is known to be avascular and the follicle is vascularised via the thecal cell layer, little is known about the role of hypoxia or HIFs in regulating ovarian function. In this study, we hypothesized that hypoxia as well as non-hypoxic stimuli cooperate in promoting follicle differentiation and luteinization via HIF activity and resultant gene regulation. We quantitatively measured the HIF1α protein response to hCG in ovarian granulosa cell cultures and in vivo and developed a transgenic (HRE(4)-SV40-EGFP) HIF reporter mouse line. We observed a time-dependent increase of HIF1α protein levels in granulosa cells post-hCG in vivo, maximal around time of ovulation. hCG alone was unable to promote HIF1α protein accumulation in cultured granulosa cells, but increased protein abundance was observed when combined with a hypoxic stimulus. HRE-EGFP ovaries showed no follicular EGFP in stages prior to antrum formation. However, HIF regulated EGFP was maximally induced in granulosa cells around the time of ovulation and readily observed in corpora lutea. There was also an increase in HIF regulated EGFP activity in the corpora lutea from functional to regressing stages. Taken together, these observations establish the notion that HIFs play a role during follicular differentiation and luteinization.