Abstract
Phospholipase D2 (PLD2) is involved in cytoskeletal reorganization, cell migration, cell cycle progression, transcriptional control and vesicle trafficking. There is no evidence about PLD2 function in oocytes during meiosis. Herein, we analyzed PLD2 expression and its relationship with spindle formation and positioning in mouse oocyte meiosis. High protein level of PLD2 was revealed in oocytes by Western blot, which remained consistently stable from prophase I with intact germinal vesicle (GV) up to metaphase II (MII) stage. Immunofluorescence showed that PLD2 appeared and gathered around the condensed chromosomesafter germinal vesicle breakdown (GVBD), and co-localized with spindle from pro-metaphase I (pro-MI) to metaphase I (MI) and at MII stage. During anaphase I (Ana I) to telophase I (Tel I) transition, PLD2 was concentrated in the spindle polar area but absent from the midbody. In oocytes incubated with NFOT, an allosteric and catalytic inhibitor to PLD2, the spindle was enlarged and center-positioned, microtubules were resistant to cold-induced depolymerization and, additionally, the meiotic progression was arrested at MI stage. However, spindle migration could not be totally prevented by PLD2 catalytic specific inhibitors, FIPI and 1-butanol, implying at least partially, that PLD2 effect on spindle migration needs non-catalytic domain participation. NFOT-induced defects also resulted in actin-related molecules’ distribution alteration, such as RhoA, phosphatidylinosital 4, 5- biphosphate (PIP2), phosphorylated Colifin and, consequently, unordered F-actin dynamics. Taken together, these data indicate PLD2 is required for the regulation of microtubule dynamics and spindle migration toward the cortex in mammalian oocytes during meiotic progression.
Highlights
Meiotic maturation of oocyte is characterized by two rounds of accurate segregations of genome and asymmetric divisions of cytoplasm, resulting in two tiny polar bodies and one large oocyte
Upon germinal vesicle breakdown (GVBD), as the chromatin was condensed into individual chromosomes (Fig. 1C, 5), Phospholipase D2 (PLD2) began to aggregate around the condensing chromosomes, simultaneously with the newly formed microtubules (Fig. 1C, 6–8)
As cell cycle progressed to pro-metaphase I and MI stage, microtubules were gradually organized into bi-polar spindle with all the chromosomes properly aligned on the equatorial plate, PLD2 sustained its co-localization with microtubules throughout the whole process of spindle organizing (Fig. 1C, 10–12, 14–16)
Summary
Meiotic maturation of oocyte is characterized by two rounds of accurate segregations of genome and asymmetric divisions of cytoplasm, resulting in two tiny polar bodies and one large oocyte. How to cite this article Liu et al (2017), PLD2 regulates microtubule stability and spindle migration in mouse oocytes during meiotic division. Spindle migration is an actin-dependent event rather than a microtubule-associated mechanism. Microtubule depolymerization with nocodazole treatment did not affect the faster cortex-towards movement of chromosomes (Li et al, 2008); either the stabilization of actin polymerization by jasplakinolide or its depolymerization by cytochalasin B impeded the migration of the spindle and the chromosomes group to the cortex (Sathananthan et al, 2006; Li et al, 2008)
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