Expression and subcellular localization of FMNL2 during mouse oocyte meiosis.

(A) Western blotting results of FMNL2 protein expression at different stages. FMNL2 expressed at the GV, MI, and MII stages. (B) Subcellular localization of FMNL2-EGFP during mouse oocyte meiosis. FMNL2 was enriched at the cortex (GV, GVBD and MI stage) and spindle periphery (MI stage). Green, FMNL2-EGFP; blue, DNA. Negative control: Green, EGFP; blue, DNA. Bar =20 μm. (C) Co-staining of oocytes for FMNL2 and actin. FMNL2 and actin overlapped in cortex and spindle surrounding. Green, FMNL2-antibody; red, actin; blue, DNA. Bar =20 μm.

Knockdown of FMNL2 affects first polar body extrusion and asymmetric division.

(A) Western blot analysis for FMNL2 expression in the FMNL2-KD group and control group. Relative intensity of FMNL2 and tubulin was assessed by densitometry. **, significant difference (P < 0.01). (B) DIC images of control oocytes and FMNL2-KD oocytes after 12 h culture. FMNL2-KD caused large polar bodies (black arrows) and some oocytes failed to extrude the polar bodies (white arrows). (C) Rate of polar body extrusion after 12 h culture of the control group and FMNL2-KD group. ***, significant difference (P < 0.001). (D) Rate of large polar body extrusion after 12 h culture in the control group and FMNL2-KD group. ****, significant difference (P < 0.0001). (E) Time-lapse microscopy showed that polar body extrusion failed after FMNL2-KD. Bar = 10 μm. (F) Western blot analysis for FMNL2 expression in the control group, FMNL2-KD group and rescue group. Relative intensity of FMNL2 and tubulin was assessed by densitometry. (G) DIC images of FMNL2-KD oocytes and rescue oocytes after 12 h culture. (H) Rate of polar body extrusion after 12 h culture of the FMNL2-KD group and rescue group. *, significant difference (P < 0.05). (I) Rate of large polar body extrusion after 12 h culture in the FMNL2-KD group and rescue group. ***, significant difference (P < 0.001). (J) Rate of polar body extrusion after 12 h culture of the control group and FMNL2+3-KD group. *, significant difference (P < 0.05). (K) Rate of large polar body extrusion after 12 h culture in the control group and FMNL2+3-KD group. ***, significant difference (P < 0.001).

Knockdown of FMNL2 disrupts spindle localization during mouse oocyte meiosis.

(A) Time-lapse microscopy showed that spindle migration failed after FMNL2-KD. Green, tubulin-EGFP. Bar = 10 μm. (B) Representative images and the proportion of spindle migration after 9.5 h of culture in the control group and FMNL2-KD oocyte group. White, actin; green, tubulin; magenta, DNA. Bar = 10 μm. *, significant difference (P < 0.05). (C) Representative images and the proportion of spindle migration after 9.5 h of culture in the FMNL2-KD group and rescue oocyte group. magenta, DNA. Bar = 10 μm. *, significant difference (P < 0.01).

Knockdown of FMNL2 disrupts actin assembly during mouse oocyte meiosis.

(A) Representative images of actin distribution at the oocyte cortex and the fluorescent intensities in the control group and FMNL2-KD group (P > 0.1). White, actin; green, tubulin; magenta, DNA. Bar = 10 μm. (B) Representative images of actin distribution in the oocyte cytoplasm and the fluorescent intensities in the control group and FMNL2-KD group. White, actin; green, tubulin; magenta, DNA. Bar = 10 μm. ****, significant difference (P < 0.0001). (C) Representative images of actin distribution in the oocyte cytoplasm and the fluorescent intensities in the FMNL2-KD group and rescue group. White, actin; magenta, DNA. Bar = 10 μm. ****, significant difference (P < 0.0001) (D) Mass spectrometry results showed that FMNL2 was related to many actin-related proteins. (E) Co-IP results showed that FMNL2 was correlated with Arp and Formin2 but not with Profiling and Fascin. (F) Arp2 protein expression significantly increased in the FMNL2-KD oocytes compared with the control oocytes. Arp2 protein expression significantly decreased in the rescue oocytes compared with the FMNL2-KD oocytes. **, significant difference (P < 0.01). (G) Formin2 protein expression significantly decreased in the FMNL2-KD oocytes compared with the control oocytes. Formin2 protein expression significantly increased in the rescue oocytes compared with the FMNL2-KD oocytes. **, significant difference (P < 0.01).

FMNL2 regulates endoplasmic reticulum distribution during mouse oocytes maturation.

(A) Co-IP results showed that FMNL2 was correlated with INF2. (B) Mass spectrometry results showed that FMNL2 was associated with ER-related proteins. (C) Representative images of ER distribution in the oocyte cytoplasm in the control group and FMNL2-KD group. In FMNL2-KD oocytes, ER agglomerated in cytoplasm (white arrow). Red, ER; Blue, DNA. Bar = 10 μm. (D) Abnormal distribution of ER significantly increased in the FMNL2-KD oocytes compared with the control oocytes. *, significant difference (P < 0.05). (E) Grp78 and Chop protein expression significantly increased in the FMNL2-KD oocytes compared with the control oocytes. The band intensity analysis also confirmed this finding. *, significant difference (P < 0.05). (F) Representative images of ER distribution in the oocyte cytoplasm in the FMNL2-KD group and rescue group. Red, ER; Blue, DNA. Bar = 10 μm. (G) Abnormal distribution of ER significantly decreased in the rescue oocytes compared with the FMNL2-KD oocytes. *, significant difference (P < 0.05). (H) Grp78 protein expression significantly decreased in the rescue oocytes compared with the FMNL2-KD oocytes. The band intensity analysis also confirmed this finding. **, significant difference (P < 0.01).

FMNL2 regulates mitochondrial distribution during mouse oocytes maturation.

(A) Mass spectrometry results showed that FMNL2 was related to many mitochondria-related proteins. (B) Representative images of mitochondrial distribution in the oocyte cytoplasm in the control group and FMNL2-KD group. In FMNL2-KD oocytes, mitochondrial agglomerated in cytoplasm (white arrow). Green, Mito. Bar = 20 μm. (C) Abnormal distribution of mitochondrial significantly increased in the FMNL2-KD oocytes compared with the control oocytes. *, significant difference (P < 0.05). (D) Representative images of mitochondrial distribution in the oocyte cytoplasm in the FMNL2-KD group and rescue group. In FMNL2-KD oocytes, mitochondrial agglomerated in cytoplasm (white arrow). Red, Mito; Blue, DNA. Bar = 20 μm. (E) Abnormal distribution of mitochondrial significantly decreased in the rescue oocytes compared with the FMNL2-KD oocytes. **, significant difference (P < 0.01). (F) The typical picture for JC1 green channel and red channel after FMNL2-KD. (G) The JC1 signal (red/green ratio) after FMNL2-KD compare with the control group, the JC-1 red/green fluorescence ratio was significantly reduced in FMNL2-KD groups. Bar = 20 µm. **, P < 0.01. (H) cofilin protein expression significantly decreased in the FMNL2-KD oocytes compared with the control oocytes. The band intensity analysis also confirmed this finding. *, significant difference (P < 0.05).