HIF1A regulates follicular atresia through O-GlcNAcylation-mediated VEZF1/ET-1/FOXO1/BAX signaling in porcine granulosa cells

Hypoxic stimuli induce follicular atresia by regulating granulosa cell (GC) apoptosis. Notably, mature follicles can still develop and ovulate under hypoxic conditions, highlighting the importance of the hypoxic adaptation in ovarian follicular selection. To date, the role and mechanism of hypoxia‐inducible factor 1 subunit alpha (HIF1A)-mediated hypoxic responses in follicular atresia are unclear. This study aimed to investigate whether and how HIF1A regulates follicular atresia via the modulation of O-linked N-acetylglucosamine (O-GlcNAc) protein modification (O-GlcNAcylation). Our findings revealed that HIF1A was highly expressed in pig ovaries. Compared with that in healthy follicles, its expression was significantly downregulated in atretic follicles. Under hypoxic conditions, pharmacological inhibition or siRNA-mediated knockdown of HIF1A increased porcine GC apoptosis. Mechanistically, HIF1A knockdown Suppressed O-GlcNAc transferase degradation, leading to increased global O-GlcNAcylation. Using 4D label-free quantitative proteomics, we identified 53 O-GlcNAcylated proteins. Importantly, O-GlcNAcylation stabilized vascular endothelial zinc finger 1 (VEZF1), and HIF1A knockdown upregulated VEZF1 protein levels by promoting O-GlcNAcylation. The HIF1A-VEZF1 axis modulates forkhead box O1 (FOXO1) expression by regulating endothelin-1. As a transcription factor, FOXO1 directly binds to the Bcl-2 associated X (BAX) promoter, activating its transcription and ultimately inducing porcine GC apoptosis and follicular atresia. Overall, our study elucidates a novel molecular mechanism by which HIF1A deficiency modulates follicular atresia through O-GlcNAcylation-mediated VEZF1 expression. These results not only clarify the molecular mechanism of ovarian follicular development under hypoxic conditions but also offer potential targets for improving follicular selection efficiency in pig breeding.