154 The involvement of hypoxia-inducible factor 1 α in bovine granulosa cell function and corpus luteum formation
K. Abbasi Samie A , M. Kowalewski A and D. Scarlet A BA Institute of Veterinary Anatomy, Vetsuisse Faculty, Zurich, Switzerland
B Clinic of Reproductive Medicine, Vetsuisse Faculty, Zurich, Switzerland
Reproduction, Fertility and Development 35(2) 205-205 https://doi.org/10.1071/RDv35n2Ab154
Published: 5 December 2022
© 2023 The Author(s) (or their employer(s)). Published by CSIRO Publishing on behalf of the IETS
Due to the avascular nature of the ovarian follicle, granulosa cell (GC) proliferation, steroidogenesis, and luteinisation occur under hypoxic conditions. Hypoxia upregulates GC steroidogenesis via activating hypoxia-inducible factor 1 α (HIF1α) and its downstream genes. Furthermore, recent research has revealed a mediating role for HIF1α in autophagy during formation of corpus luteum (CL) in rats. Thus, the objective of this study was to analyse the expression of genes involved in bovine GC function and CL formation using an in vitro model. Ovaries were collected from a local slaughterhouse and cells were seeded on six-well plates to grow to 60–70% confluence and pre-incubated under different O2 conditions: normoxia (20%) or reduced O2 tension (10% in a hypoxic chamber) for 24 h. Afterwards, cells were stimulated under the same O2 conditions with 100 ng/mL IGF1 for 24 h, the time point associated with the highest STAR and progesterone output in pilot experiments. In blocking experiments, cells were pre-incubated with 5 nM echinomycin (Ech; functional blocker of HIF1a) for 40 min. All experiments were repeated four times. GraphPad software was used for statistical analysis with one-way analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparison post hoc test. Expression of STAR was three-fold increased (P < 0.05) by stimulation with IGF1, and this effect was inhibited by Ech. Reduced O2 content did not have an effect on STAR expression. Blocking of HIF1α with Ech did not affect (P > 0.05) the expression of autophagy-related genes, BNIP3 and BECN1, independent of O2 tension. In contrast, the anti-apoptotic gene BCL2 was less abundant (P < 0.05) after stimulation with IGF1 and after blocking with Ech under both 20% and 10% O2. Furthermore, inhibition of HIF1α with Ech increased two-fold (P < 0.001) FGF2 abundance in GC in comparison with control and IGF1 treatment, independent of O2 content, and this effect was highest (P < 0.05) under 10% O2. Similarly, blocking of HIF1α with Ech increased eight-fold (P < 0.05) COX2 transcript, regardless of O2 tension. Cumulatively, our results implicate enhanced apoptosis (decreased expression of the anti-apoptotic gene BCL2) as well as GC proliferation and survival (increased expression of the pro-angiogenic and cell proliferation inducing factor FGF2 together with the rate-limiting enzyme in the conversion of arachidonic acid into prostaglandins COX2) in response to the functional suppression of HIF1α, regardless of O2 concentration (20% vs 10%). BCL2 is known to interact with BECN1 to control cell haemostasis through autophagy. It binds to BNIP3 to prevent its impact on cell death, playing functional roles during CL formation. Under our experimental conditions, BECN1 and BNIP3 were not affected (P > 0.05) by Ech. Further studies are needed to understand the autophagy-related molecular mechanisms underlying the involvement of HIF1α and reduced O2 in regulating GC function and luteinisation.
