Fam70A binds Wnt5a to regulate meiosis and quality of mouse oocytes
Fam70A binds Wnt5a to regulate meiosis and quality of mouse oocytes
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Fam70A 结合 Wnt5a 调节小鼠卵母细胞减数分裂和质量
DOI：
10.1111/cpr.12825
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发表时间：
2020-05-11
影响因子：
8.5
通讯作者：
Zhang, Dong
中科院分区：
文献类型：
--
作者：
Zhang, Na-Na;Zhang, Teng;Zhang, Dong
目的卵母细胞减数分裂中除通道、载体和受体外的膜整合蛋白的作用尚不清楚。跨膜蛋白Fam 70 A先前被鉴定为Fox 3a敲除小鼠卵巢中可能的“雌性生育因子”，其中几乎所有卵泡都经历同步激活，并且小鼠很早就变得不育。然而，Fam 70 A是否在卵母细胞减数分裂中起作用仍不清楚。材料与方法采用免疫共沉淀法、免疫金标记电镜法、共定位法和酵母双杂交法等方法对两种蛋白的相互作用进行了验证。使用抗体或小干扰RNA转染来耗尽蛋白质。免疫荧光、免疫组织化学和活体示踪剂染色用于检查定位或表征表型。结果Fam 70 A在卵母细胞膜上富集，对正常减数分裂有重要作用。Fam 70 A缺失显著破坏纺锤体组装、染色体聚集和第一极体排出，从而增加非整倍体和异常受精。此外，Fam 70 A直接结合Wnt 5a，卵母细胞内最丰富的Wnt成员。Fam 70 A或Wnt 5a的消耗显著增加腺瘤性结肠息肉病（APC），其稳定活性β-连环蛋白和微管。因此，Fam 70 A或Wnt 5a的耗尽显著增加了β-连环蛋白（非活性形式）和乙酰化微管蛋白，而APC敲除显著降低了这两者。结论Fam 70 A通过经典和非经典Wnt 5a信号通路调控小鼠卵母细胞减数分裂和质量。
Objectives Little is known about the roles of integral membrane proteins beyond channels, carriers or receptors in meiotic oocytes. The transmembrane protein Fam70A was previously identified as a likely "female fertility factor" in Fox3a-knockout mouse ovaries where almost all follicles underwent synchronous activation and the mice became infertile very early. However, whether Fam70A functions in oocyte meiosis remains unknown. Therefore, the present study aimed to address this question.Materials and Methods Co-immunoprecipitation, immunogold labelling-electron microscopy, co-localization and yeast two-hybrid assays were used to verify the interaction. Antibody or small interfering RNA transfection was used to deplete the proteins. Immunofluorescence, immunohistochemistry and live tracker staining were used to examine the localization or characterize phenotypes. Western blot was used to examine the protein level.Results Fam70A was enriched in oocyte membranes important for normal meiosis. Fam70A depletion remarkably disrupted spindle assembly, chromosome congression and first polar body extrusion, which subsequently increased aneuploidy and abnormal fertilization. Moreover, Fam70A directly bound Wnt5a, the most abundant Wnt member within oocytes. Depletion of either Fam70A or Wnt5a remarkably increased adenomatous polyposis coli (APC), which stabilizes active beta-catenin and microtubules. Consequently, depletion of either Fam70A or Wnt5a remarkably increased p-beta-catenin (inactive form) and acetylated tubulin, while APC knockdown remarkably decreased these two. Furthermore, Fam70A depletion remarkably reduced Akt phosphorylation.Conclusions Fam70A regulates meiosis and quality of mouse oocytes through both canonical and non-canonical Wnt5a signalling pathways.