Rab32-based vesicles coordinate mitochondria and actin for spindle migration and organelle rearrangement in oocyte meiosis

Abstract

Introduction: Rab32 is a part of the Rab GTPase family, which is known as the regulator of vesicle transport for an array of cellular functions including endosomal transport, autophagy, generation of melanosomes, phagocytosis and inflammatory processes.

Objective: However, the role of Rab32 in oocyte meiosis is still not well-defined.

Methods: We depleted Rab32 expression by knock down approach, and we also disrupted Rab32 function by exogenous Rab32^Q83L/T37N mRNA injection for mutation.

Results: In our current investigation, we delved into its impacts on the cytoskeleton dynamics and the functionality of organelles during the meiotic maturation process in mouse oocytes. Rab32 expressed during oocyte meiosis and deletion of Rab32 or the expression of exogenous Rab32^Q83L/T37N led to oocyte polar body extrusion defects or symmetric division. We showed that Rab32 was essential for ROCK1-based actin assembly which further led to spindle migration for the asymmetry. Besides, perturbation of Rab32 affected DRP1 phosphorylation for the spatial arrangement and functionality of mitochondria in mouse oocytes. And we found that Rab32 disruption caused the miscarriage of membrane organelles such as Golgi apparatus, ER, lysosome and CGs during oocyte meiosis, leading to ER stress and autophagy.

Conclusions: In summary, our study unravels the critical functions of Rab32 for the interplay between actin and mitochondria, which further facilitates movement of the spindle apparatus and organelles arrangement in mouse oocyte meiotic development.

Keywords: Actin; Meiosis; Mitochondria; Organelles; Rab32.

Graphical abstract

Fig. 1

Expression and subcellular distribution of Rab32. (A) The expression levels of Rab32 at GV, GVBD, MI, MII stages were detected by western blotting in mouse oocytes. (B) Subcellular localization of Rab32 was marked in mouse oocytes. Rab32 was widely distributed in the cytoplasm. Red, Rab32; Green, tubulin; blue, DNA; scale bar, 20 μm. (C) The exogenous Rab32 exhibited a broad distribution within the cytoplasm. Red, Myc-Rab32; Green, tubulin; blue, DNA; scale bar, 20 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Disruption of Rab32 function impairs oocyte maturation. (A) mRNA level of Rab32 was counted after Rab32-siRNA injection. Control (n = 30), Rab32-KD (n = 30), 1 vs. 0.18 ± 0.05. (B) Rab32 protein level was examined by western blot after Rab32 siRNA injection. The band intensities were analyzed by Image J software. 1 vs. 0.43 ± 0.04. (C) Rab32 defects caused the failure of polar body extrusion and proportion of large polar body. scale bar, 20 μm. (D) The statistical analysis of the percentage of polar body extrusion. Control (n = 129), Rab32-KD (n = 141), polar body extrusion: 67.35 ± 2.51 % vs. 46.10 ± 1.01 %, Cohen’s d = 5.54, 95 % CI [1.74, 9.35]. (E) The statistical analysis of large polar body rates. Control (n = 129), Rab32-KD (n = 141), large polar body: 16.03 ± 3.52 % vs. 44.80 ± 2.89 %, Cohen’s d = 5.15, 95 % CI [0.44, 9.86]. (F) Injection of Rab32 Q83L and Rab32 T37N caused the failure of polar body extrusion, accompanied by a slight asymmetric division. scale bar, 20 μm. (G) A decline of polar body extrusion rates after overexpression of GTP- or GDP-bound Rab32. Control (n = 154), Rab32 Q83L (n = 136), Rab32 T37N (n = 142), polar body extrusion: control group: 83.27 ± 4.22 %, vs. Rab32 Q83L overexpression group: 61.13 ± 5.567 %, Cohen’s d = 2.59, 95 % CI [0.49, 5.66] vs. Rab32 T37N overexpression group, 54.93 ± 2.521 %, Cohen’s d = 4.71, 95 % CI [0.31, 9.11]; large polar body: control group: 0.63 ± 0.63 %, n = 154 vs. Rab32 Q83L overexpression group: 4.40 ± 0.67 %, Cohen's d = 3.35, 95 % CI [0.17, 6.86] vs. Rab32 T37N overexpression group: 5.90 ± 0.12 %, Cohen's d = 6.70, 95 % CI [0.87, 12.49]. * P < 0.05, ** P < 0.01.

Fig. 3

Rab32 regulates meiotic spindle migration during oocyte maturation. (A) The spindle stayed in the center of the oocyte after Rab32 knockdown instead of migrating to the cortex. Green, tubulin; blue, DNA; scale bar, 20 μm. The statistical analysis of spindle migration. Control (n = 135), Rab32-KD (n = 137), 67.80 ± 3.31 % vs. 37.57 ± 3.45 %. (B) Deletion of Rab32 decreased the cytoplasmic actin around the spindle. Red, actin; blue, DNA; scale bar, 20 μm. The statistical analysis of cytoplasmic actin fluorescence intensity showed significantly reduced after Rab32 knockdown. Control (n = 135), Rab32-KD (n = 137), 1 vs. 0.71 ± 0.06. (C) Quantitative analysis of the extent of spindle migration indicated that the L/D ratio was significantly higher after injection of Rab32 Q83L and Rab32 T37N. Control (n = 93), Rab32 Q83L (n = 97), Rab32 T37N (n = 93), control group: 1 vs. Rab32 Q83L: 1.06 ± 0.17 vs. Rab32 T37N, 1.17 ± 0.01. Pink, actin; Green, spindle. (D) The cytoplasmic actin enhanced in the Rab32 Q83L mRNA injection group, and Rab32 T37N mRNA injection decreased cytoplasmic actin. Red, actin; green, spindle; blue, DNA; scale bar, 20 μm. The statistical analysis of the cytoplasmic actin fluorescence intensity after overexpressing Rab32. Control (n = 93), Rab32 Q83L (n = 97), Rab32 T37N (n = 93), control group: 1 vs. Rab32 Q83L: 1.15 ± 0.03 vs. Rab32 T37N, 0.87 ± 0.02. (E) Co-IP results showed that Rab32 was correlated with ROCK1. (F) ROCK1 expression levels after overexpression of Rab32 Q83L or Rab32 T37N, and the quantification of western bolt bands. Control group: 1 vs. Rab32 Q83L overexpression group: 1.36 ± 0.04 vs. Rab32T37N overexpression group, 0.53 ± 0.02. (G) The reduction of Rab32 led to decreased localization of ROCK1 around the spindle. Control (n = 15), Rab32-KD (n = 14), 26630 ± 523.90 vs. 24693 ± 546.70. (H) Overexpression of Rab32 Q83L mRNA elevated the intensity of ROCK1 at the periphery of spindle, while Rab32 T37N overexpression diminished ROCK1. Control (n = 15), Rab32 Q83L (n = 14), Rab32 T37N (n = 14), control group: 34582 ± 560.30, vs. Rab32 Q83L: 36042 ± 297.00 vs. Rab32 T37N, 30445 ± 594.80. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and ns, no significant difference. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Rab32 maintains mitochondrial function in mouse oocyte meiosis. (A) Injection Rab32 siRNA caused aberrant mitochondrial distribution. Red, mitochondria; blue, DNA; scale bar, 20 μm. (B) The fluorescence intensity analysis of mitochondria showed a significant descend after Rab32 knocking down. Control (n = 73), Rab32-KD (n = 69), 1, vs. 0.55 ± 0.08. (C) TMRE was used to mark the mitochondrial membrane potential (MMP) showing an obvious decline in Rab32 depletion. Red, TMRE; blue, DNA; scale bar, 20 μm. (D) The statistical analysis of MMP fluorescence intensity showing with a significant decrease on the Rab32-KD group. Control (n = 75), Rab32-KD (n = 76), 1 vs. 0.60 ± 0.05. (E) The relative content of mtDNA copy number in the Rab32 siRNA-injected group was apparent reduced. Control (n = 200), Rab32-KD (n = 200), 1 vs. 0.80 ± 0.03. (F) The relative content of ATP level in the Rab32 siRNA-injected group was significantly decreased. Control (n = 90), Rab32-KD (n = 90), 1 vs. 0.58 ± 0.05. (G) Fluo-4 AM was used to label the calcium level in mitochondria showing with a decline of calcium level in Rab32-KD oocytes. Green, calcium; blue, DNA; scale bar, 20 μm. (H) The statistical analysis of the calcium fluorescence intensity, and Rab32 knockdown caused significantly weakening. Control (n = 75), Rab32-KD (n = 78), 1 vs. 0.55 ± 0.10. (I) Compared with the control group, injection of GTP-bound Rab32 induced mitochondria remarkably clustered around the spindle, and injection of GDP-bound Rab32 induced mitochondria dispersed to cytoplasm. Red, mitochondria; blue, DNA; scale bar, 20 μm. (J) The statistical analysis of the aberrant mitochondrial distribution, and it elevated sharply in the overexpression groups. Control (n = 72), Rab32 Q83L (n = 68), Rab32 T37N (n = 69), control group: 25.10 ± 3.69 % vs. Rab32 Q83L: 51.10 ± 3.19 % vs. Rab32 T37N, 56.00 ± 1.10 %. (K) Western blot results showed p-DRP1 expression levels after overexpression of Rab32 Q83L or Rab32T37N, and the quantification of western bolt bands. Control group: 1 vs. Rab32 Q83L overexpression group: 0.79 ± 0.02 vs. Rab32 T37N overexpression group, 0.69 ± 0.08. * P < 0.05, ** P < 0.01, *** P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Rab32 regulates Golgi-based cortical granule transport during oocyte maturation. (A) The accumulation of Golgi around the spindle was reduced with an abnormal distribution. Green, Golgi; blue, DNA; scale bar, 20 μm. (B) The statistical analysis of Golgi abnormal distribution, and Rab32-KD significantly disturbed Golgi localization and the relative fluorescence intensity analysis of Golgi. Control (n = 87), Rab32-KD (n = 90), abnormal Golgi: 28.33 ± 1.67 % vs. 58.57 ± 8.00 %; fluorescence intensity: 1 vs. 0.83 ± 0.02. (C) Injection of Rab32 Q83L led to the evenly distribution of Golgi into cytoplasm, while injection of Rab32 T37N resulted in slight agglutination of Golgi in the cytoplasm. Green, Golgi; blue, DNA; scale bar, 20 μm. (D) The statistical analysis of Golgi abnormal distribution. It demonstrated injection of Rab32 Q83L disturbed Golgi localization, and injection of Rab32 T37N had no significant effects on Golgi distribution. Control (n = 42), Rab32 Q83L (n = 39), Rab32 T37N (n = 44), control group: 27.08 ± 2.08 % vs. Rab32 Q83L: 63.57 ± 2.85 % vs. Rab32 T37N, 34.20 ± 2.27 %, no significance. (E) Western blot results showed GM130 expression levels after overexpression of Rab32 Q83L or Rab32T37N and the quantification of western bolt results. Control group: 1 vs. Rab32 Q83L: 0.73 ± 0.07 vs. Rab32 T37N, 0.74 ± 0.02. (F) Injection of Rab32 siRNA led to errors in the transport of cortical granules (CGs). Green, CGs; blue, DNA; scale bar, 20 μm. (G) The graph showed a failure of CGs migration to the cortex and the related statistical analysis. Control (n = 73), Rab32-KD (n = 79), 72.90 ± 0.20 % vs. 49.00 ± 4.06 %. (H) The cortical granules failed to migrated properly to the cortex and form an empty area after injection of GTP- and/or GDP-bound Rab32 mRNA. Green, CGs; blue, DNA; scale bar, 20 μm. (I) The statistical analysis of CGs aberrant migration rate. Control (n = 54), Rab32 Q83L (n = 56), Rab32 T37N (n = 45), control group: 27.23 ± 2.11 % vs. Rab32 Q83L: 45.73 ± 2.40 % vs. Rab32 T37N, 68.38 ± 3.25 %. * P < 0.05, ** P < 0.01, **** P < 0.0001, and ns, no significant difference. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Rab32 regulates ER and lysosome distribution during oocyte meiosis. (A) The ER around spindle declined after Rab32 knocking down, and floated to the cytoplasm. Green, ER; blue, DNA; scale bar, 20 μm. (B) The statistical analysis of ER abnormal distribution, and Rab32-KD significantly disturbed ER localization. And the relative fluorescence intensity analysis of ER showed decreased after Rab32 siRNA injection. Control (n = 87), Rab32-KD (n = 90), abnormal ER: 18.90 ± 1.10 % vs. 60.40 ± 3.53 %; fluorescence intensity:1 vs. 0.46 ± 0.07. (C) Overexpression of GTP-bound Rab32 induced aberrant aggregation of ER in cytoplasmic, and overexpression of GDP-bound Rab32 caused contracts area of ER around the spindle. Green, ER; blue, DNA; scale bar, 20 μm. (D) The statistical analysis of ER abnormal distribution, showing with higher abnormal rates. Control (n = 60), Rab32 Q83L (n = 55), Rab32 T37N (n = 57), control group: 19.40 ± 3.96 % vs. Rab32 Q83L: 47.00 ± 3.57 % vs. Rab32 T37N: 50.00 ± 2.59 %. (E) Western blot results showed LC3 and GRP78 expression levels after overexpression of Rab32 Q83L or Rab32 T37N. (F) Quantification analysis of western bolt results. GRP78: control group: 1 vs. Rab32 Q83L: 0.57 ± 0.02 vs. Rab32 T37N: 1.67 ± 0.10; LC3: control group: 1 vs. Rab32 Q83L: 1.28 ± 0.13 vs. Rab32T37N, 1.52 ± 0.40, no significance. (G) Rab32-KD induced an aggregated lysosome mass in cytoplasm. Red, lysosome; blue, DNA; scale bar, 20 μm. (H) The statistical analysis of lysosome abnormal distribution. (I) Overexpression of Rab32 resulted in excessive agglutination and/or decreased lysosomal vesicles in oocyte. Control (n = 57), Rab32-KD (n = 52), 19.83 ± 1.63 % vs. 73.43 ± 5.50 %. Red, lysosome; blue, DNA; scale bar, 20 μm. (J) The statistical analysis of lysosome aberrant distribution. Control (n = 59), Rab32 Q83L (n = 56), Rab32 T37N (n = 57), control group: 27.13 ± 3.13 % vs. Rab32 Q83L: 59.87 ± 3.14 % vs. Rab32 T37N, 53.63 ± 2.05 %. * P < 0.05, ** P < 0.01, *** P < 0.001, and ns, no significant difference. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

Roles of Rab32 during mouse oocyte maturation. Schematic representation of Rab32 regulating the crosstalk between mitochondria and actin for spindle migration and organelles rearrangement during oocyte meiotic maturation.

Supplementary Fig. S1

Antibody specificity validation. (A) The full-length band of endogenous Rab32. (B) The localization of Rab32 staining with Rab32 antibody after overexpression Rab32 mutants. (C) The immunofluorescence images of Rab38, which indicated the localization of Rab38 associated with the spindle. (D) The separate staining experiment of the secondary antibody, serving as a negative control to demonstrate the specificity of the Rab32 antibody. (E) The target recognition site of mouse monoclonal anti-Rab32 antibody, which was distinct from Rab38 and Rab7.

Supplementary Fig. S2

(A) Rab32 defects had no significant influence on cortical actin. Red, actin; blue, DNA; scale bar, 20 μm. (B) The statistical analysis of the fluorescence intensity of cortical actin, it showed no significance between control and Rab32-KD groups. Control (n = 135), Rab32-KD (n = 137), 1 vs. 0.95 ± 0.04, no significance. (C) Overexpression of Rab32 had no significant effects on cortical actin. Gray, actin; blue, DNA; scale bar, 20 μm. (D) The statistical analysis of the cortical actin after overexpressing of Rab32 showing with no difference. Control (n = 93), Rab32 Q83L (n = 97), Rab32 T37N (n = 93), control group: 1 vs. Rab32 Q83L: 0.92 ± 0.04, no significance vs. Rab32 T37N, 1.02 ± 0.04, no significance.