Jiang Aiwen, Li Jialong, Wang Luyao, Liu Yi, Wu Zhengchang, Wang Haifei, Wu Shenglong, Bao Wenbin
Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design, College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, China.
Joint International Research Laboratory of Agriculture & Agri-Product Safety, Yangzhou University, Yangzhou, 225009, China.
J Anim Sci Biotechnol. 2025 Sep 20;16(1):127. doi: 10.1186/s40104-025-01263-0.
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.
缺氧刺激通过调节颗粒细胞（GC）凋亡诱导卵泡闭锁。值得注意的是，成熟卵泡在缺氧条件下仍可发育并排卵，这突出了缺氧适应在卵巢卵泡选择中的重要性。迄今为止，缺氧诱导因子1α亚基（HIF1A）介导的缺氧反应在卵泡闭锁中的作用和机制尚不清楚。本研究旨在探讨HIF1A是否以及如何通过调节O-连接的N-乙酰葡糖胺（O-GlcNAc）蛋白修饰（O-GlcNAcylation）来调节卵泡闭锁。
我们的研究结果显示，HIF1A在猪卵巢中高表达。与健康卵泡相比，其在闭锁卵泡中的表达显著下调。在缺氧条件下，HIF1A的药理学抑制或siRNA介导的敲低增加了猪颗粒细胞凋亡。机制上，HIF1A敲低抑制了O-GlcNAc转移酶的降解，导致整体O-GlcNAcylation增加。使用4D无标记定量蛋白质组学，我们鉴定了53种O-GlcNAcylated蛋白。重要的是，O-GlcNAcylation稳定了血管内皮锌指1（VEZF1），并且HIF1A敲低通过促进O-GlcNAcylation上调了VEZF1蛋白水平。HIF1A-VEZF1轴通过调节内皮素-1来调节叉头框O1（FOXO1）的表达。作为一种转录因子，FOXO1直接结合到Bcl-2相关X蛋白（BAX）启动子上，激活其转录并最终诱导猪颗粒细胞凋亡和卵泡闭锁。
总体而言，我们的研究阐明了一种新的分子机制，即HIF1A缺乏通过O-GlcNAcylation介导的VEZF1表达来调节卵泡闭锁。这些结果不仅阐明了缺氧条件下卵巢卵泡发育的分子机制，也为提高猪育种中卵泡选择效率提供了潜在靶点。
