摘要Background 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 adap-tation 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).Results Our findings revealed that HIF1A was highly expressed in pig ovaries.Compared with that in healthy fol-licles,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 knock-down 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-GIcNAcylation.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.Conclusion 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.