Luo Hui, Chen Jianhua, Li Cao, Wu Tian, Yin Siyue, Yang Guangping, Wang Yipin, Guo Zhihan, Hu Saifei, He Yanni, Wang Yingnan, Chen Yao, Su Youqiang, Miao Congxiu, Qian Yun, Feng Ruizhi
State Key Laboratory of Reproductive Medicine and Offspring Health, Nanjing Medical University, Nanjing, China.
Yangzhou Maternal and Child Health Care Hospital Affiliated to Yangzhou University, Yangzhou, China.
Clin Transl Med. 2025 Jan;15(1):e70193. doi: 10.1002/ctm2.70193.
Numerous pathogenic variants causing human oocyte maturation arrest have been reported on the primate-specific TUBB8 gene. The main etiology is the dramatic reduction of tubulin α/β dimer, but still large numbers of variants remain unexplained.
Using microinjection mRNA and genome engineering to reintroduce the conserved pathogenic missense variants into oocytes or in generating TUBB8 variant knock-in mouse models, we investigated that the human deleterious variants alter microtubule nucleation and spindle assembly during meiosis. Live-cell imaging and immunofluorescence were utilised to track the dynamic expression of microtubule plus end-tracking proteins in vivo and analysed microtubule nucleation or spindle assembly in vitro, respectively. Immunoprecipitation-mass spectrometry and ultramicro-quantitative proteomics were performed to identify the differential abundance proteins and affected interactome of TUBB8 protein.
First, we observed a significant depletion of the EB1 signal upon microinjection of mutated TUBB8 mRNA (including R262Q, M300I, and D417N missense variants), indicating disruption of microtubule nucleation caused by these introduced TUBB8 missense variants. Mechanically, we demonstrated that the in vivo TUBB8-D417N missense variant diminished the affinity of EB1 and microtubules. It also harmed the interaction between microtubules and CKAP5/TACC3, which are crucial for initiating microtubule nucleation. Attenuated Ran-GTP pathway was also found in TUBB8-D417N oocytes, leading to disrupted spindle assembly. Stable microtubule was largely abolished on the spindle of TUBB8-D417N oocytes, reflected by reduced tubulin acetylation and accumulated HDAC6. More importantly, selective inhibition of HDAC6 by culturing TUBB8-D417N oocytes with Tubacin or Tubastatin A showed morphologically normal spindle and drastically recovered polar-body extrusion rate. These rescue results shed light on the strategy to treat meiotic defects in a certain group of TUBB8 mutated patients.
Our study provides a comprehensive mechanism elucidating how TUBB8 missense variants cause oocyte maturation arrest and offers new therapeutic avenues for treating female infertility in the clinic.
灵长类动物特有的TUBB8基因上已报道了许多导致人类卵母细胞成熟停滞的致病变异。主要病因是微管蛋白α/β二聚体的显著减少，但仍有大量变异无法解释。
我们通过显微注射mRNA和基因组工程将保守的致病性错义变异重新引入卵母细胞或构建TUBB8变异敲入小鼠模型，研究人类有害变异如何在减数分裂过程中改变微管成核和纺锤体组装。利用活细胞成像和免疫荧光分别在体内追踪微管正端追踪蛋白的动态表达以及在体外分析微管成核或纺锤体组装。进行免疫沉淀-质谱分析和超微量定量蛋白质组学以鉴定差异丰度蛋白和TUBB8蛋白受影响的相互作用组。
首先，我们观察到显微注射突变的TUBB8 mRNA（包括R262Q、M300I和D417N错义变异）后EB1信号显著减少，表明这些引入的TUBB8错义变异导致微管成核破坏。从机制上讲，我们证明体内TUBB8-D417N错义变异降低了EB1与微管的亲和力。它还损害了微管与CKAP5/TACC3之间的相互作用，而这对启动微管成核至关重要。在TUBB8-D417N卵母细胞中还发现Ran-GTP途径减弱，导致纺锤体组装破坏。TUBB8-D417N卵母细胞纺锤体上的稳定微管基本消失，表现为微管蛋白乙酰化减少和HDAC6积累。更重要的是，用Tubacin或Tubastatin A培养TUBB8-D417N卵母细胞对HDAC6的选择性抑制显示纺锤体形态正常且极体排出率大幅恢复。这些挽救结果为治疗某一组TUBB8突变患者的减数分裂缺陷提供了策略。
我们的研究提供了一个全面的机制，阐明了TUBB8错义变异如何导致卵母细胞成熟停滞，并为临床治疗女性不孕症提供了新的治疗途径。
