Department of Histology and Embryology, School of Basic Medical Sciences, Capital Medical University, Beijing, China.
Department of Human Reproductive Medicine, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing, China.
Autophagy. 2024 Jul;20(7):1616-1638. doi: 10.1080/15548627.2024.2333164. Epub 2024 Mar 27.
PLD1 has been implicated in cytoskeletal reorganization and vesicle trafficking in somatic cells; however, its function remains unclear in oocyte meiosis. Herein, we found PLD1 stably expresses in mouse oocytes meiosis, with direct interaction with spindle, RAB11A vesicles and macroautophagic/autophagic vacuoles. The genetic or chemical inhibition of PLD1 disturbed MTOC clustering, spindle assembly and its cortical migration, also decreased PtdIns(4,5)P, phosphorylated CFL1 (p-CFL1 [Ser3]) and ACTR2, and their local distribution on MTOC, spindle and vesicles. Furthermore in PLD1-suppressed oocytes, vesicle size was significantly reduced while F-actin density was dramatically increased in the cytoplasm, the asymmetric distribution of autophagic vacuoles was broken and the whole autophagic process was substantially enhanced, as illustrated with characteristic changes in autophagosomes, autolysosome formation and levels of ATG5, BECN1, LC3-II, SQSTM1 and UB. Exogenous administration of PtdIns(4,5)P or overexpression of CFL1 hyperphosphorylation mutant (CFL1) could significantly improve polar MTOC focusing and spindle structure in PLD1-depleted oocytes, whereas overexpression of ACTR2 could rescue not only MTOC clustering, and spindle assembly but also its asymmetric positioning. Interestingly, autophagy activation induced similar defects in spindle structure and positioning; instead, its inhibition alleviated the alterations in PLD1-depleted oocytes, and this was highly attributed to the restored levels of PtdIns(4,5)P, ACTR2 and p-CFL1 (Ser3). Together, PLD1 promotes spindle assembly and migration in oocyte meiosis, by maintaining rational levels of ACTR2, PtdIns(4,5)P and p-CFL1 (Ser3) in a manner of modulating autophagy flux. This study for the first time introduces a unique perspective on autophagic activity and function in oocyte meiotic development.: ACTR2/ARP2: actin related protein 2; ACTR3/ARP3: actin related protein 3; ATG5: autophagy related 5; Baf-A1: bafilomycin A; BFA: brefeldin A; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GOLGA2/GM130: golgin A2; GV: germinal vesicle; GVBD: germinal vesicle breakdown; IVM: maturation; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MI: metaphase of meiosis I; MII: metaphase of meiosis II; MO: morpholino; MTOC: microtubule-organizing center; MTOR: mechanistic target of rapamycin kinase; PB1: first polar body; PLA: proximity ligation assay; PLD1: phospholipase D1; PtdIns(4,5)P/PIP2: phosphatidylinositol 4,5-bisphosphate; RAB11A: RAB11A, member RAS oncogene family; RPS6KB1/S6K1: ribosomal protein S6 kinase B1; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TUBA/α-tubulin: tubulin alpha; TUBG/γ-tubulin: tubulin gamma; UB: ubiquitin; WASL/N-WASP: WASP like actin nucleation promoting factor.
PLD1 已被牵连到体细胞中的细胞骨架重组和囊泡运输中；然而，其在卵母细胞减数分裂中的功能仍然不清楚。在此，我们发现 PLD1 在小鼠卵母细胞减数分裂中稳定表达，与纺锤体、RAB11A 囊泡和巨自噬/自噬小体直接相互作用。PLD1 的遗传或化学抑制扰乱了 MTOC 聚集、纺锤体组装及其皮质迁移，还降低了 PtdIns(4,5)P、磷酸化的 CFL1（Ser3）和 ACTR2，以及它们在 MTOC、纺锤体和囊泡上的局部分布。此外，在 PLD1 抑制的卵母细胞中，囊泡大小显著减小，而细胞质中 F-肌动蛋白密度显著增加，自噬小体的不对称分布被打破，整个自噬过程显著增强，自噬小体、自噬溶酶体形成和 ATG5、BECN1、LC3-II、SQSTM1 和 UB 的水平都有特征性变化。外源性给予 PtdIns(4,5)P 或过表达磷酸化突变体（CFL1）可显著改善 PLD1 耗竭卵母细胞中的极性 MTOC 聚焦和纺锤体结构，而过表达 ACTR2 不仅可以拯救 MTOC 聚集和纺锤体组装，还可以拯救其不对称定位。有趣的是，自噬激活诱导了类似的纺锤体结构和定位缺陷；相反，其抑制缓解了 PLD1 耗竭卵母细胞的改变，这主要归因于 PtdIns(4,5)P、ACTR2 和 p-CFL1（Ser3）水平的恢复。总的来说，PLD1 通过维持 ACTR2、PtdIns(4,5)P 和 p-CFL1（Ser3）的合理水平，以调节自噬流的方式，促进卵母细胞减数分裂中的纺锤体组装和迁移。这项研究首次介绍了自噬活性和功能在卵母细胞减数分裂发育中的独特视角。
