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 (CFL1^S3E) 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.

Keywords: Autophagy; MTOC clustering; PLD1; meiosis; oocyte; spindle migration.

Figure 1.

Expression and subcellular localization of PLD1 in mouse oocytes during meiosis. (A) Western blot analysis detected stable expression of PLD1. Each sample contained 90 cells which were collected after 0, 2, 8 and 16 h in vitro maturation (IVM) culture, corresponding to GV, GVBD, MI and MII. (B) Representative immunofluorescence images showed PLD1 subcellular localization at GV, GVBD, MI, telophase I (tel I) and MII. Upon GVBD, PLD1 began to colocalize with the reassembled microtubules around chromosomes. Along with the cell cycle to MII, PLD1 overlapped with microtubules in the spindle apparatus. Arrowheads indicated dot-like aggregations of PLD1 in cytoplasm; arrows indicated PLD1 on spindle. Red, PLD1; green, acetylated-TUBA/α-tubulin (tubulin alpha); blue, DNA. Scale bar: 20 μm. (C) Representative immunofluorescence images illustrated PLD1 colocalization with vesicles in oocytes. Arrowheads indicated dot-like aggregations of PLD1 and GOLGA2 in cytoplasm. Red, PLD1; green, GOLGA2; blue, DNA. Scale bar: 20 μm.

Figure 2.

PLD1 depletion suppressed spindle migration and assembly in oocyte meiosis. (A-i) Western blot analysis of PLD1 protein in oocytes of the uninjected group, Ctrl MO group and Pld1 MO group. Each sample had 90 oocytes. (A-ii) quantitative analysis of PLD1 protein level in oocytes of uninjected group, Ctrl MO group and Pld1 MO group. (B) MYC protein level of oocytes with different microinjection treatments by western blot analysis. Each sample had 50 oocytes. (C) Representative images of oocytes in Ctrl MO group, Pld1 MO group and Pld1 MO + Myc-Pld1 group at 8 h of IVM. Arrowheads indicated dot-like congression of TUBG on spindle poles. Red, TUBG; green, acetylated-TUBA; blue, DNA. Scale bar: 20 μm. (D) computational method for measurement of spindle size and migration distance. (E-K) statistical analysis of oocytes with asymmetrical spindle (%), relative area of spindle (%), spindle length (l/R), width of chromosome region (d2/R), oocytes with defocused polar MTOC (%), numbers of polar MTOC foci per oocyte and spindle migration (d1/R) in Ctrl MO (n = 116), Pld1 MO (n = 137) and Pld1 MO + Myc-Pld1 (n = 120) groups. All data were presented as the mean percentage (mean ± SEM) of at least three independent experiments. ***p < 0.001, ****p < 0.0001 by ordinary one-way ANOVA analysis.

Figure 3.

PLD1 modulated PB1 volume and asymmetric division in mouse oocytes. (A) Representative images of oocytes in DMSO group and VU groups at 16 h of IVM, respectively. Green, acetylated-TUBA; blue, DNA. Scale bar: 20 μm. (B) Representative images of oocytes in DMSO group and VU group at 16 h of IVM, respectively. Black arrows showed oocytes with large PB1. Scale bar: 400 μm. (C, D) statistical analysis of relative volumn of PB1(%) and MII rate (%) in DMSO group (n = 146) and 5 μM (n = 149), 7.5 μM (n = 136) and 10 μM (n = 140) VU-treated groups. (E) Representative images of oocytes in ctrl MO group, Pld1 MO group and Pld1 MO + myc-Pld1 group at 16 h of IVM, respectively. Green, acetylated-TUBA; blue, DNA. Scale bar: 20 μm. (F) Representative images of oocytes in ctrl MO group, Pld1 MO group and Pld1 MO + myc-Pld1 group at 16 h of IVM, respectively. Black arrows showed oocytes with large PB1. Scale bar: 400 μm. (G, H) statistical analysis of relative volumn of PB1(%) and MII rate (%) in ctrl MO group (n = 128), Pld1 MO group (n = 120) and Pld1 MO + myc-Pld1 group (n = 114). All data were presented as the mean percentage (mean ± SEM) of at least three independent experiments. ***p < 0.05, ****p < 0.0001 by ordinary one-way ANOVA analysis.

Figure 4.

PLD1 depletion disturbed vesicle fusion and F-actin density in mouse oocytes. (A) Representative images of oocytes in ctrl MO group, Pld1 MO group and Pld1 MO + myc-Pld1 group at 8 h of IVM, respectively. Arrowheads indicated patch-like aggregations of GOLGA2 and RAB11A in cytoplasm. Red, RAB11A; green, GOLGA2; blue, DNA. Scale bar: 20 μm. (B, C) statistical analysis of numbers of large RAB11A-positive vesicles (>1000 pixels) and small RAB11A-positive vesicles (<1000 pixels) in ctrl MO group (n = 112), Pld1 MO group (n = 116) and Pld1 MO + myc-Pld1 group (n = 111). (D, G) Representative transmission electron microscopy (TEM) images of oocytes in ctrl MO group, Pld1 MO group and Pld1 MO + myc-Pld1 group at 8 h of IVM, respectively. Scale bar: 500 nm. (E, F) statistical analysis of numbers of large vesicles (maximum diameter >0.2 μm) and small vesicles (maximum diameter 0.1 μm-0.2 μm) per 3 µm × 3 µm area in ctrl MO group (n = 4), Pld1 MO group (n = 4) and Pld1 MO + myc-Pld1 group (n = 4). (H, I) statistical analysis of numbers of golgi apparatus fragment and percentage of abnormal golgi apparatus fragment (%) in ctrl MO group (n = 4), Pld1 MO group (n = 4) and Pld1 MO + myc-Pld1 group (n = 4). (J) Representative images of oocytes in ctrl MO group, Pld1 MO group and Pld1 MO + myc-Pld1 group at 8 h of IVM, respectively. Arrow indicated actin cap on cytoplasmic membrane. Green, F-actin; blue, DNA. Scale bar: 20 μm. (K) statistical analysis of F-actin fluorescence intensity (AU) in ctrl MO group (n = 40), Pld1 MO group (n = 34) and Pld1 MO + myc-Pld1 group (n = 35). All data were presented as the mean percentage (mean ± SEM) of at least three independent experiments. ***p < 0.05, ****p < 0.0001 by ordinary one-way ANOVA analysis.

Figure 5.

Direct physical interaction between PLD1 and vesicle proteins. (A-i) Representative images of PLA signals in different groups. PLA showed the proximity in distance of PLD1 and GOLGA2 in oocytes. Scale bar: 20 μm. (A-ii) quantitative analysis of PLA signal dots in different groups. >100 pixels dots were involved in the analysis. Data were presented as the mean percentage (mean ± SEM) of at least three independent experiments. PLD1 Rb ab group: n = 100; GOLGA2 ms ab group: n = 100; GOLGA2 ms ab + RAB11A Rb ab group: n = 102; Ctrl MO – GOLGA2 ms ab + PLD1 Rb ab group: n = 107; Pld1 MO – GOLGA2 ms ab + PLD1 Rb ab group: n = 105. (B) Co-IP was performed to determine the direct interaction between PLD1 and GOLGA2, RAB11A. Oocytes lysates were incubated with IgG or anti-PLD1 antibody, respectively. The blots of IP eluates were probed with anti-PLD1, anti-GOLGA2 and anti-RAB11A antibodies, respectively. (C-i) expression of GOLGA2 and RAB11A in oocytes in Ctrl MO group and Pld1 MO group by western blot. Each sample had 80 oocytes. (C-ii and iii) quantitative analysis of protein level changes in oocytes. All data were presented as the mean percentage (mean ± SEM) of at least three independent experiments. ***p < 0.0001 by ordinary one-way ANOVA analysis. p > 0.05 by unpaired t test.

Figure 6.

Both regulatory and catalytic region of PLD1 were required in spindle assembly and migration. (A) MYC-protein level of oocytes with vehicle, Pld1^F120A,R179Q and Pld1^K898R cRNA microinjection treatments by western blot analysis. Each sample had 50 oocytes. (B) Representative images of MI oocytes in vehicle, Pld1^F120A,R179Q and Pld1^K898R groups, respectively. Arrowheads indicated signal of TUBG on spindle poles. Red, TUBG; green, acetylated-TUBA; blue, DNA. Scale bar: 20 μm. (C-I) statistical analysis of spindle migration (d1/R), relative area of spindle (%), spindle length (l/R), asymmetrical spindle (%), width of chromosome region (d2/R), oocytes with defocused polar MTOC (%) and polar MTOC foci per oocyte in vehicle (n = 113), Pld1^F120A,R179Q (n = 102) and Pld1^K898R (n = 101) groups. (J) Representative images of MII oocytes in vehicle, Pld1^F120A,R179Q and Pld1^K898R groups, respectively. Green, acetylated-TUBA; blue, DNA. Scale bar: 20 μm. (K) Representative images of MII oocytes in vehicle, Pld1^F120A,R179Q and Pld1^K898R groups, respectively. Black arrows showed oocytes with large PB1. Scale bar: 400 μm. (L, M) statistical analysis of relative volumn of PB1(%) and MII rate (%) in vehicle (n = 100), Pld1^F120A,R179Q (n = 103) and Pld1^K898R (n = 102) groups. (N) Representative images of MI oocytes in vehicle, Pld1^F120A,R179Q and Pld1^K898R groups, respectively. Arrowheads indicated patch-like aggregations of GOLGA2 and RAB11A in cytoplasm. Red, RAB11A; green, GOLGA2. Scale bar: 20 μm. (O, P) statistical analysis of numbers of large RAB11A-positive vesicles (>1000 pixels) and small RAB11A-positive vesicles (<1000 pixels) in vehicle (n = 115), Pld1^F120A,R179Q (n = 105) and Pld1^K898R (n = 110) groups. All data were presented as the mean percentage (mean ± SEM) of at least three independent experiments. ***p < 0.001, ****p < 0.0001 by ordinary one-way ANOVA analysis.

Figure 7.

PLD1 depletion decreased PtdIns(4,5)P2, p-CFL1(Ser3) and ACTR2. (A) changes of protein expression in oocytes in ctrl MO group and Pld1 MO group by western blot. The blots were incubated with anti-FMN2, anti-PLD1, anti-PtdIns(4,5)P₂, anti-ACTR3, anti-ACTR2, anti-CFL1, anti-p-CFL1(Ser3) and anti-GAPDH antibodies, respectively. Each sample had 90 oocytes. (B – H) quantitative analysis of protein level changes in oocytes. (I) changes of protein expression in oocytes in groups of ctrl MO, Pld1 MO and Pld1 MO + myc-Pld1 by western blot. The blots were incubated with anti-PtdIns(4,5)P₂, anti-ACTR2, anti-p-CFL1 (Ser3) and anti-GAPDH antibodies, respectively. Each sample had 90 oocytes. (J-L) quantitative analysis of protein level changes in oocytes. (M) Representative images of MI oocytes in ctrl MO, Pld1 MO and Pld1 MO+ myc-Pld1 groups. Arrowheads indicated foci of signal of ACTR2 and PtdIns(4,5)P₂. Red, p-CFL1 (Ser3); green, ACTR2 or PtdIns(4,5)P₂; blue, DNA. Scale bar: 20 μm. (N, O) statistical analysis of numbers of large ACTR2 dots (>1000 pixels) and small ACTR2 dots (<1000 pixels) in groups of ctrl MO (n = 102), Pld1 MO (n = 99) and Pld1 MO + myc-Pld1 (n = 102) groups. (P, Q) statistical analysis of oocytes with defocused PtdIns(4,5)P₂ (%) and numbers of PtdIns(4,5)P₂ dots in poles in groups of ctrl MO (n = 102), Pld1 MO (n = 99) and Pld1 MO + myc-Pld1 (n = 102). (R, S) statistical analysis of oocytes with defocused polar p-CFL1 (Ser3) (%) and numbers of p-CFL1 (Ser3) dots in poles in groups of ctrl MO (n = 102), Pld1 MO (n = 99) and Pld1 MO + myc-Pld1 (n = 102). (T) Co-IP was performed to determine the interaction between PLD1 and PtdIns(4,5)P₂, ACTR2 and CFL1. Oocytes lysates were incubated with IgG or anti-PLD1 antibody. The blots of IP eluates were probed with anti-PLD1, anti-PtdIns(4,5)P₂, anti-ACTR2 and anti-CFL1 antibodies, respectively. All data were presented as the mean percentage (mean ± SEM) of at least three independent experiments. ***p < 0.01, ****p < 0.001,****p < 0.0001 by unpaired t test or ordinary one-way ANOVA analysis.

Figure 8.

CFL1^S3E and exogenous PtdIns(4,5)P2 can rescue MTOC fragmentation caused by PLD1 depletion or inhibition. (A) Western blot analysis of MYC-protein level in oocytes injected with vehicle, Cfl1^S3A and Cfl1^S3E cRNA. Each sample contained 50 oocytes. (B) Representative images of MI oocytes in groups of Ctrl, Cfl1^S3A, Pld1 MO and Pld1 MO + Cfl1^S3E, respectively. Arrowheads indicated signal of TUBG on spindle poles. Red, TUBG; green, acetylated-TUBA; blue, DNA. Scale bar: 20 μm. (C-I) statistical analysis of spindle migration (d1/R), relative area of spindle (%), spindle length (l/R), oocytes with asymmetrical spindle (%), width of chromosome region (d2/R), defocused polar MTOC (%) and polar MTOC foci per oocyte in groups of ctrl (n = 125), Cfl1^S3A (n = 123), Pld1 MO (n = 124) and Pld1 MO + Cfl1^S3E (n = 129). (J) Representative images of MI oocytes in DMSO, 5 μM VU and VU + PtdIns(4,5)P₂ groups. Arrowheads indicated signal of TUBG on spindle poles. Red, TUBG; green, acetylated-TUBA; blue, DNA. Scale bar: 20 μm. (K-Q) statistical analysis of spindle migration (d1/R), relative area of spindle (%), spindle length (l/R), width of chromosome region (d2/R), oocytes with asymmetrical spindle (%), defocused polar MTOC (%) and polar MTOC foci per oocyte in different groups. DMSO group: n = 120, 5 μM VU group: n = 114, VU +10 μM PtdIns(4,5)P₂ group: n = 118, VU +15 μM PtdIns(4,5)P₂ group: n = 116, VU +20 μM PtdIns(4,5)P₂ group: n = 121, VU +25 μM PtdIns(4,5)P₂ group: n = 118. All data were presented as the mean percentage (mean ± SEM) of at least three independent experiments. ***p < 0.05, ****p < 0.01, ****p < 0.001,***p < 0.0001 by ordinary one-way ANOVA analysis.

Figure 9.

ACTR2 overexpression reversed the failures in MTOC focusing, spindle assembly and migration in PLD1-depleted oocytes. (A) MYC-protein level in oocytes injected with vehicle and myc-Actr2 cRNA by western blot analysis. Each sample had 50 oocytes. (B) Representative images of MI oocytes in groups of ctrl MO, Pld1 MO and Pld1 MO + myc-Actr2, respectively. Arrowheads indicated concentration of TUBG on spindle poles. Red, TUBG; green, acetylated-TUBA; blue, DNA. Scale bar: 20 μm. (C-I) statistical analysis of spindle migration (d1/R), relative area of spindle (%), spindle length (l/R), width of chromosome region (d2/R), oocytes with asymmetrical spindle (%), oocytes with defocused polar MTOC (%) and numbers of MTOC dots in poles in different groups. ctrl MO group: n = 113, Pld1 MO group: n = 115, Pld1 MO + myc-Actr2 group: n = 116. (J) statistical analysis of relative size of PB1(%) in ctrl MO (n = 103), Pld1 MO (n = 100) and Pld1 MO + myc-Actr2 (n = 100) groups. (K) Representative images of MI oocytes in groups of ctrl MO, Pld1 MO and Pld1 MO + myc-Actr2. Arrowheads indicated aggregations of GOLGA2 and RAB11A in cytoplasm. Red, RAB11A; green, GOLGA2; blue, DNA. Scale bar: 20 μm. (L, M) statistical analysis of numbers of large RAB11A-positive vesicles (>1000 pixels) and small RAB11A-positive vesicles (<1000 pixels) in different groups. ctrl MO group: n = 114, Pld1 MO group: n = 116, Pld1 MO+ myc-Actr2 group: n = 118. All data were presented as the mean percentage (mean ± SEM) of at least three independent experiments. ***p < 0.01, ****p < 0.001, ****p < 0.0001 by ordinary one-way ANOVA analysis.

Figure 10.

PLD1 depletion enhanced autophagy. (A) Representative transmission electron microscopy (TEM) images of MI oocytes in groups of ctrl MO, Pld1 MO and Pld1 MO + myc-Pld1, respectively.Yellow arrowheads showed autophagosomes, red arrowheads showed autolysosomes. Scale bar: 500 nm. (B) statistical analysis of numbers of autolysosomes in ctrl MO (n = 4), Pld1 MO (n = 4) and Pld1 MO + myc-Pld1 (n = 4) groups. (C-i) expression of MTOR, p-MTOR (S2448) , RPS6KB1, p-RPS6KB (T389), SQSTM1, LC3-II, ATG5 and BECN1 in oocytes in ctrl MO and Pld1 MO group by western blot. Each sample had 80 oocytes. (C-ii-vii) quantitative analysis of protein level changes in oocytes. (D) Co-IP was performed to determine the interaction between PLD1 and SQSTM1, LC3. Oocytes lysates were incubated with IgG or anti-PLD1 antibody. The blots of IP eluates were probed with anti-PLD1, anti-SQSTM1 and anti-LC3 antibodies, respectively. (E) expression of PtdIns(4,5)P₂, SQSTM1, ACTR2, p-CFL1 (Ser3) and LC3 in oocytes in groups of ctrl MO, baf-A1, Pld1 MO and Pld1 MO + baf-A1 by western blot. Each sample had 80 oocytes. (F-J) quantitative analysis of protein level changes in oocytes. (K) Representative images of MI oocytes in groups of ctrl MO, baf-A1, Pld1 MO and Pld1 MO + baf-A1, respectively. Arrowheads indicated patch-like aggregations of LC3 in cytoplasm; the dashed line displayed the regional specificity of autophagy signal distribution. Red, LC3; green, SQSTM1; blue, DNA. Scale bar: 20 μm. (L) quantitative analysis of oocytes with asymmetrical distribution of SQSTM1 and LC3(%). ctrl MO group: n = 101, baf-A1 group: n = 111, Pld1 MO group: n = 121, Pld1 MO + baf-A1 group: n = 116. All data were presented as the mean percentage (mean ± SEM) of at least three independent experiments. ***p < 0.05, ****p < 0.01, ****p < 0.001, ***p < 0.0001, by unpaired t test or ordinary one-way ANOVA analysis.

Figure 11.

Autophagy inhibition could rescue spindle assembly and migration failure caused by PLD1 depletion. (A) Representative images of MTOC (red), spindle (green) and chromosomes (blue) in MI oocytes from groups of ctrl MO, baf-A1, Pld1 MO and Pld1 MO + baf-A1 groups, respectively. Arrowheads indicated signal of TUBG on spindle poles. Scale bar: 20 μm. (B-H) statistical analysis indicates difference in spindle migration (d1/R), relative area of spindle (%), spindle length (l/R), oocytes with asymmetrical spindle (%), width of chromosome region (d2/R), oocytes with defocused polar MTOC (%), numbers of MTOC dots in poles and asymmetrical spindle among different groups. ctrl MO group: n = 120, baf-A1 group: n = 115, Pld1 MO group: n = 113, Pld1 MO + baf-A1 group: n = 120. (I) Representative images of vesicles in MI oocytes from groups of ctrl MO, baf-A1, Pld1 MO and Pld1 MO + baf-A1, respectively. Arrowheads displayed the aggregations of GOLGA2 and RAB11A. Red, RAB11A; green, GOLGA2; blue, DNA. Scale bar: 20 μm. (J, K) statistical analysis of numbers of large RAB11A-positive vesicles (>1000 pixels) and small RAB11A-positive vesicles (<1000 pixels) in different groups. ctrl MO group: n = 120, baf-A1 group:n = 120, Pld1 MO group: n = 114, Pld1 MO + baf-A1 group: n = 120. (L) Representative images of F-actin in MI oocytes from different groups. Arrow indicated actin cap on cytoplasmic membrane. Green, F-actin; blue, DNA. Scale bar: 20 μm. (M) statistical analysis of F-actin fluorescence intensity (AU) in different groups. ctrl MO group: n = 32, baf-A1 group: n = 31, Pld1 MO group: n = 33, Pld1 MO + baf-A1 group: n = 32. All data were presented as the mean percentage (mean ± SEM) of at least three independent experiments. ***p < 0.01, ****p < 0.0001 by ordinary one-way ANOVA analysis.

Figure 12.

A schematic diagram depicting roles of PLD1 in mouse oocyte meiosis. Left panel, in the presence of PLD1, autophagy is maintained at rational level, spindle assembly and cortical migration orderly occur in oocyte meiosis. Right panel, in the absence of PLD1, autophagy is hyperfunctional, spindle assembly and migration are destroyed by defective changes in MTOC clustering, vesicle fusion and F-actin assembly, due to reduction in ACTR2, PtdIns(4,5)P₂ and p-CFL1 (Ser3).