Oxygen Depletion and HIF1α Regulate Steroidogenesis in Mouse Granulosa Cells by Modulating Cholesterol Metabolism and Lipid Availability

Olivia E. Smith¹, Luiz A. Berto Gomes¹, Sybilla Rzaca¹, Mariusz P. Kowalewski¹

¹Institute of Veterinary Anatomy, Vetsuisse Faculty, University of Zurich, CH-8057 Zurich, Switzerland.

Abstract Text: The avascular environment within growing ovarian follicles creates low-oxygen (O₂) conditions that support granulosa cell function and oocyte development. Hypoxia-Inducible Factor (HIF)1α, a master regulator of biological responses to hypoxia, plays a crucial role as a transcription factor in controlling steroid production. It has both direct and indirect effects on cAMP-mediated, STAR-dependent steroidogenesis. In mouse granulosa cells, STAR protein levels are higher under acute exposure to reduced oxygen tension (10% O₂, moderate hypoxia, for 6h), compared to 20% O₂ (normoxia). However, severe O₂ reduction (1% O₂ for 6h) decreases STAR expression in these cells, indicating that the degree of O₂ deprivation can impact granulosa cell function. The molecular mechanisms underlying steroidogenesis under oxygen deprivation, as well as the potential role of the naturally lowered O₂ tension (physoxia) remain unclear. To investigate this, transcriptomic (RNA-Seq) and proteomic (LC-MS/MS) analyses were performed using immortalized murine granulosa cells(KK1). Differentially expressed genes (DEGs) and proteins (DEPs) were identified (Transcriptomics: P< 0.05, FDR < 0.01; Proteomics: minimum peptide number 2, FDR < 0.1) to evaluate how HIF1α and chronic (30h) exposure to reduced O₂ influence cAMP-stimulated steroidogenesis. The involvement of HIF1α was assessed using its specific inhibitor, Echinomycin. In moderate and severe hypoxia (2488 and 7461 DEGs, respectively), the expression of genes involved in steroid metabolism, cAMP/PKA signaling, and responses to hypoxia, was significantly altered. Interestingly, while proteomic analysis measured reduced expression of STAR protein in severe hypoxia, supporting previous findings, transcriptomic analysis revealed a downregulation of Star in samples cultured under both reduced O₂ conditions, i.e. moderate and severe hypoxia. Concomitantly, the transcriptional expression of Hif1α was also downregulated in both hypoxic environments, together with the upregulation of HIF1α regulators Egln1 (PHD2), Egln3 (PHD3) and Vhl, known to induce its proteasomal degradation. This activation of HIF1α regulation in chronic hypoxic environments suggests compensatory mechanisms that modulate HIF1α expression and, thereby, its availability, presumably preventing the negative effects of HIF1α overexpression implicated in earlier studies. Other notable pathways affected by chronic O₂ reduction included cholesterol biosynthesis, intracellular transport, and mitochondrial function. These transcriptional shifts were mirrored at the proteomic level, where analysis of the 328 differentially expressed proteins (DEPs) under severe hypoxia underscored the importance of intracellular cholesterol transport and steroid metabolism, affecting key proteins such as ABCA1, SQLE, LIPE (HSL) and LDLR. Accordingly, after blocking HIF1α activity, both transcriptomics and proteomics revealed significant disruptions in lipid and fatty acid metabolism and cholesterol transfer activity. Next, building on transcriptomic and proteomic observations, the size and number of lipid droplets (LD) were assessed in cells stained with the BODIPY™ 493/503 fluorophore. While cAMP alone generally reduced LD size within the oxygenation groups, indicating greater cholesterol utilization in stimulated cells, an increase in LD size was observed in cells stimulated under both moderate and severe hypoxia compared to normoxia. This suggests enhanced cholesterol storage in low O₂ conditions. Notably, echinomycin reversed the reducing effect of cAMP on LD size, restoring it to levels comparable to unstimulated controls. This further highlights the essential role of HIF1α in supplying cholesterol during the acute steroidogenic response. Our findings provide a foundational understanding of the molecular adaptations to altered O₂ availability, emphasize the importance of HIF1α, and offer insights relevant to both physiological conditions in steroidogenic cells, and pathological states in which dysregulated steroid production is a hallmark. (Supported by SNSF grant nr:310030_207895)