Fig 2: HIF1 is essential for granulosa cell function. Expression of HIF1A is induced by FSH, IGF1 and low oxygen levels in bovine granulosa cells. HIF1 may regulate steroidogenesis, cell proliferation, angiogenesis and inflammation processes in developing ovarian follicles.

Fig 3: Effects of HIF1A knockdown on cell proliferation. (a,b) depicts CCND2 and PCNA mRNA expressions upon HIF1A knockdown in granulosa cells under normoxic and hypoxic conditions. (c,d) depict the percent of replicating cells (S-phase) under normoxic and hypoxic conditions, respectively, upon treating with control and HIF1A gapmers. Data were represented in the mean ± SEM values of three independent culture experiments (n = 3). Significant differences were acknowledged at the minimum level of p < 0.05 by t-test.

Fig 4: Immunohistochemical evaluation of letrozole-treated chorioallantoises (CA). (A) Immunohistochemical demonstration of capillary densities in chorioallantoic membranes of letrozole-treated and control mares using vWF. The capillary density tended to be greater in the control CAs than in the letrozole-treated CAs, p = 0.07; (B) A strong cytoplasmic signal for VEGF is noted within the chorionic epithelium of letrozole-treated group. No difference was observed in the abundance of HIF1A+ cells between the groups. (B) Red boxes (letrozole) and blue circles (control) represent values for the capillary density scores in the chorioallantoic samples.

Fig 5: Effects of echinomycin on candidate gene expression. Suppression of HIF1 activity using echinomycin altered the mRNA expression of HIF1A (a), VEGFA (b) and VNN2 (c) genes under normoxic and hypoxic conditions. Data were presented in Mean ± SEM values of three independent cell culture experiments (n = 3). Significant differences were acknowledged at the minimum level of p < 0.05 by one way repeated measures analysis of variance. Pairwise comparisons were analyzed using Post hoc Tuckey test.