PLD1 promotes spindle assembly and migration through regulating autophagy in mouse oocyte meiosis.

ABSTRACT

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)P2, 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)P2 or overexpression of CFL1 hyperphosphorylation mutant (CFL1S3E) 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)P2, ACTR2 and p-CFL1 (Ser3). Together, PLD1 promotes spindle assembly and migration in oocyte meiosis, by maintaining rational levels of ACTR2, PtdIns(4,5)P2 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.Abbreviations ACTR2/ARP2 actin related protein 2; ACTR3/ARP3 actin related protein 3; ATG5 autophagy related 5; Baf-A1 bafilomycin A1; BFA brefeldin A; GAPDH glyceraldehyde-3-phosphate dehydrogenase; GOLGA2/GM130 golgin A2; GV germinal vesicle; GVBD germinal vesicle breakdown; IVM in vitro 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)P2/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.