Abstract：
Spindle migration and assembly regulates asymmetric oocyte division, which is essential for fertility. Fbxo28, as a member of SCF (Skp1-Cul1-F-box) ubiquitin E3 ligases complex, is specifically expressed in oocytes. However, little is known about the functions of Fbxo28 in spindle assembly and migration during oocyte meiosis I. In present study, microinjection with morpholino oligonucleotides and exogenous mRNA for knockdown and rescue experiments, and immunofluorescence staining, western blot, timelapse confocal microscopy and chromosome spreading were utilized to explore the roles of Fbxo28 in asymmetric division during meiotic maturation. Our data suggested that Fbxo28 mainly localized at chromosomes and acentriolar microtubule-organizing centers (aMTOCs). Depletion of Fbxo28 did not affect polar body extrusion but caused defects in spindle morphology and migration, indicative of the failure of asymmetric division. Moreover, absence of Fbxo28 disrupted both cortical and cytoplasmic actin assembly and decreased the expression of ARPC2 and ARP3. These defects could be rescued by exogenous Fbxo28-myc mRNA supplement. Collectively, this study demonstrated that Fbxo28 affects spindle morphology and actin-based spindle migration during mouse oocyte meiotic maturation.
摘要：
纺锤迁移和组装调节不对称卵母细胞分裂，这对生育至关重要。Fbxo28，作为SCF（Skp1-Cul1-F-box）泛素E3连接酶复合物的成员，在卵母细胞中特异性表达。然而,关于Fbxo28在卵母细胞减数分裂过程中的纺锤体组装和迁移中的功能知之甚少。在目前的研究中，吗啉代寡核苷酸和外源mRNA的显微注射用于敲除和挽救实验，免疫荧光染色，westernblot,利用延时共聚焦显微镜和染色体扩散来探索Fbxo28在减数分裂成熟过程中不对称分裂中的作用。我们的数据表明，Fbxo28主要位于染色体和子粒微管组织中心（aMTOC）。Fbxo28的耗尽不会影响极体挤出，但会导致纺锤体形态和迁移缺陷，表示不对称分裂的失败。此外，缺乏Fbxo28破坏了皮质和细胞质肌动蛋白的组装，并降低了ARPC2和ARP3的表达。这些缺陷可以通过外源性Fbxo28-mycmRNA补充来挽救。总的来说，这项研究表明，Fbxo28在小鼠卵母细胞减数分裂成熟过程中影响纺锤体形态和基于肌动蛋白的纺锤体迁移。
