Fig 2: Effect of Wapl depletion on cohesin binding.(A) Representative images of localization of Rec8 on the chromosomes in control and Wapl-MO oocytes. GV oocytes were injected with control or Wapl-specific morpholinos, maintained for 20 hours in 2.5 μM milrinone before being washed into milrinone-free medium to allow resumption of meiosis. At 6 hours after GVBD, oocytes immunostained for Rec8 and DNA (Hoechst) following chromosome spreading. Scale bar, 10 μm. (B) The fluorescence intensity of Rec8 was measured in control (n = 200, bivalents) and Wapl-MO (n = 200, bivalents) oocytes. (C) Representative images of localization of Smc3 on the chromosomes in control and Wapl-MO oocytes. GV oocytes were injected with control or Wapl-specific morpholinos, maintained for 20 hours in 2.5 μM milrinone before being washed into milrinone-free medium to allow resumption of meiosis. At 6 hours after GVBD, oocytes were immunostained for Smc3 and DNA (Hoechst) following chromosome spreading. Scale bar, 10 μm. (D) The fluorescence intensity of Smc3 was measured in control (n = 200, bivalents) and Wapl-MO (n = 200, bivalents) oocytes. (E) The protein levels of Rec8 were assessed by immunoblotting in control and Wapl-MO oocytes. Two hundred oocytes at 6 hours after GVBD for each group were collected and immunoblotted for Wapl, Rec8, and Gapdh. (F) The protein levels of Smc3 were assessed by immunoblotting in control and Wapl-MO oocytes. Two hundred oocytes at 6 hours after GVBD for each group were collected and immunoblotted for Wapl, Smc3, and Gapdh. (G) Representative images of bivalents and kinetochores in control and Wapl-MO oocytes. GV oocytes were injected with control or Wapl-specific morpholinos, maintained for 20 hours in 2.5 μM milrinone before being washed into milrinone-free medium to allow resumption of meiosis. At 6 hours after GVBD, oocytes were immunostained for CREST and DNA (Hoechst) following chromosome spreading. Scale bar, 10 μm. (H) The distance between kinetochore pairs within a bivalent was measured in control (n = 200, kinetochore pairs) and Wapl-MO (n = 200, kinetochore pairs) oocytes. Data of (B), (D), and (H) were presented as mean value (mean ± SEM) of at least three independent experiments.

Fig 3: The interaction between Wapl and Bub3.(A) Identification of binding partners of Wapl by immunoprecipitation and MS. The protein coverage, the number of peptides, and the molecular weight were shown for each molecule. (B) Co-IP was performed to determine the interaction between Wapl and Bub3. Oocytes were collected at 3 hours after GVBD (prometaphase I stage). Oocyte lysates were incubated with immunoglobulin G (IgG) and anti-Wapl antibody, respectively, followed by incubation with protein G beads. The blots of 5% of input and IP eluates were probed with anti-Bub3 and anti-Wapl antibodies, respectively. IP eluates were immunoblotted for histone H3 as a negative control. (C) Reciprocal co-IP was performed with IgG and anti-Bub3 antibody, respectively. The blots of 5% of input and IP eluates were probed with anti-Wapl and anti-Bub3 antibodies, respectively. (D) HEK293 cells were cotransfected with Wapl-Flag and Bub3-HA plasmids and subjected to co-IP using anti-Flag antibody. The interaction between Wapl-Flag and Bub3-HA was shown by the presence of Bub3-HA in the IP eluate pulled down by anti-Flag antibody. Five percent of input was used for immunoblotting. (E) Colocalization of Wapl with Bub3. At 3 hours after GVBD, oocytes were costained for Wapl and Bub3 following chromosome spreading. Scale bar, 5 μm. (F) Localization of Bub3 at prometaphase I stage in control, Wapl-MO, and Wapl-rescue oocytes. GV oocytes were injected with control or Wapl-specific morpholinos, maintained for 20 hours in 2.5 μM milrinone before being washed into milrinone-free medium to allow resumption of meiosis. For the rescue experiment, GV oocytes were injected with Wapl-specific morpholinos and maintained for 20 hours in 2.5 μM milrinone before being injected with Wapl mRNA and maintained for a further 2 hours in 2.5 μM milrinone to allow time for Wapl translation. Oocytes were then washed into milrinone-free medium to allow resumption of meiosis. At 3 hours after GVBD, oocytes were fixed and immunostained for Bub3, CREST, and DNA (Hoechst). Scale bar, 10 μm. (G) The relative fluorescence intensity of Bub3 to CREST was measured in control (n = 189), Wapl-MO (n = 183), and Wapl-rescue (n = 171) oocytes. The signal intensity of Bub3 was normalized with that of CREST. (H) The fluorescence intensity of Bub3 was measured in control (n = 189), Wapl-MO (n = 183), and Wapl-rescue (n = 171) oocytes. (I) The protein levels of Bub3 were detected by immunoblots of control, Wapl-MO, and Wapl-rescue oocytes. Two hundred oocytes at 3 hours after GVBD for each group were collected and immunoblotted for Wapl, Bub3, and α-tubulin. (J) Coomassie-stained SDS-polyacrylamide gel electrophoresis (PAGE) gel of the purified recombinant Wapl fragments and full-length Bub3. 1–300, Wapl1–300; 301–600, Wapl301–600; 601–900, Wapl601–900; 901–1200, Wapl901–1200. (K) Coomassie-stained SDS-PAGE gel of the recombinant Bub3 bound to the recombinant Wapl1–300 fragment following glutathione S-transferase (GST) pull down. (L) Coomassie-stained SDS-PAGE gel of the purified recombinant Wapl fragments. 1–50, Wapl1–50; 51–300, Wapl51–300. (M) Coomassie-stained SDS-PAGE gel of the recombinant Bub3 bound to the recombinant Wapl1–50 fragment following GST pull down. (N) HEK293 cells were cotransfected with wild-type (WT) or mutant (YSR>ASE) Wapl-Flag and Bub3-HA plasmids and then subjected to co-IP experiment using anti-Flag antibody. The interaction between Wapl-Flag and Bub3-HA was shown by the presence of Bub3-HA in the IP eluate pulled down by anti-Flag antibody. Ten percent of input was used for immunoblotting. (O) The protein levels of Bub3 were detected by immunoblotting in control, Wapl-MO, and mutant (YSR>ASE) Wapl-rescue oocytes. Two hundred oocytes at 3 hours after GVBD for each group were collected and immunoblotted for Wapl, Bub3, and α-tubulin. (P) Meiotic progression in control (n = 113), Wapl-MO (n = 110), and mutant (YSR>ASE) Wapl-rescue (n = 108) oocytes was shown by recording the rate of first PBE from 5 to 10 hours after GVBD. GV oocytes were injected with control or Wapl-specific morpholinos, maintained for 20 hours in 2.5 μM milrinone before being washed into milrinone-free medium to allow resumption of meiosis. For the rescue experiments, GV oocytes were injected with Wapl-specific morpholinos and maintained for 20 hours in 2.5 μM milrinone before being injected with mutant (YSR>ASE) Wapl mRNA and maintained for a further 2 hours in 2.5 μM milrinone to allow time for mutant Wapl translation. Oocytes were then washed into milrinone-free medium to allow resumption of meiosis. Data of (G), (H), and (P) were presented as mean value (mean ± SEM) of at least three independent experiments. ***P < 0.001.

Fig 4: Meiotic defects in Wapl-depleted oocytes could be restored by expression of exogenous Bub3.(A) Meiotic progression in control (n = 135), Wapl-MO (n = 124), and Bub3-rescue (n = 115) oocytes was determined by recording the rate of first PBE from 5 to 10 hours after GVBD. GV oocytes were injected with control or Wapl-specific morpholinos, maintained for 20 hours in 2.5 μM milrinone before being washed into milrinone-free medium to allow resumption of meiosis. For the rescue experiment, GV oocytes were injected with Wapl-specific morpholino and maintained for 20 hours in 2.5 μM milrinone before being injected with Bub3 mRNA and maintained for a further 2 hours in 2.5 μM milrinone to allow time for Bub3 translation. Oocytes were then washed into milrinone-free medium to allow resumption of meiosis. (B) Representative images of spindle morphologies and chromosome alignment in control, Wapl-MO, and Bub3-rescue oocytes. At 6 hours after GVBD, oocytes were fixed and immunostained for α-tubulin and DNA (PI). Scale bar, 20 μm. (C) Abnormal spindles with misaligned chromosomes were quantified in control (n = 75), Wapl-MO (n = 82), and Bub3-rescue (n = 87) oocytes. (D) The spindle length was measured in control (n = 37), Wapl-MO (n = 33), and Bub3-rescue (n = 38) oocytes at M I stage. (E) The width of M I plate was measured in control (n = 36), Wapl-MO (n = 38), and Bub3-rescue (n = 35) oocytes. (F) Representative images of K-MT attachment in control, Wapl-MO, and Bub3-rescued oocytes. At 6 hours after GVBD, oocytes were fixed and immunostained for α-tubulin, CREST, and DNA (Hoechst). White and yellow arrows indicate nonconnected kinetochores and polar kinetochores, respectively. Scale bar, 5 μm. (G) The rate of unattached kinetochores was recorded in control (n = 25), Wapl-MO (n = 27), and Bub3-rescue (n = 22) oocytes. (H) Representative images of euploid and aneuploid M II eggs. Chromosome spreading was performed to count the number of chromosomes in control, Wapl-MO, and Bub3-rescued oocytes at 10 hours after GVBD. Scale bar, 5 μm. (I) The rate of hyperploid eggs was recorded in control (n = 66), Wapl-MO (n = 61), and Bub3-rescue (n = 59) oocytes. Data of (A), (C), and (I) were presented as mean percentage (mean ± SEM) of at least three independent experiments. Data of (D), (E), and (G) were presented as mean value (mean ± SD) of at least three independent experiments. **P < 0.01, ***P < 0.001. (J) Working model of how Wapl functions in control of the SAC during meiosis I. In normal oocytes, Wapl binds to and stabilizes Bub3 to maintain the SAC function, consequently ensuring euploid eggs. However, in oocytes depleted of Wapl, the SAC function is compromised due to the degradation of Bub3, resulting in aneuploid eggs.

Fig 5: Effect of Wapl depletion on the spindle assembly, chromosome alignment, K-MT attachment, and euploidy in mouse oocytes.(A) Representative images of spindle morphologies and chromosome alignment in control and Wapl-MO oocytes. GV oocytes were injected with control or Wapl-specific morpholinos, maintained for 20 hours in 2.5 μM milrinone before being washed into milrinone-free medium to allow resumption of meiosis. At 6 hours after GVBD, oocytes were fixed and immunostained for α-tubulin and DNA (Hoechst). Arrowheads indicate nonaligned bivalents. Scale bar, 20 μm. (B) The rate of abnormal spindles was recorded in control (n = 102) and Wapl-MO (n = 108) oocytes. (C) The rate of apolar, elongated, multipolar, and short spindles was recorded in control (n = 102) and Wapl-MO (n = 108) oocytes. (D) The rate of misaligned chromosomes was recorded in control (n = 102) and Wapl-MO (n = 108) oocytes. (E) Representative images of the width of M I plate in control and Wapl-MO oocytes. At 6 hours after GVBD, oocytes were fixed and immunostained for α-tubulin and DNA (PI). Scale bar, 10 μm. (F) The width of M I plate was measured in control (n = 38) and Wapl-MO (n = 40) oocytes. (G) Representative images of K-MT attachment in control and Wapl-MO oocytes. At 6 hours after GVBD, oocytes were incubated in M2 medium at 4°C for 10 min to induce the depolymerization of unstable microtubules and then immediately fixed and immunostained for α-tubulin, CREST, and DNA (Hoechst). White, yellow, and green arrows indicate nonconnected kinetochores, polar kinetochores, and stretched bivalents, respectively. Scale bar, 5 μm. (H) The number of unattached kinetochores was recorded in control (n = 26) and Wapl-MO (n = 27) oocytes. (I) Representative images of euploid and aneuploid M II eggs. Chromosome spreading was performed to count the number of chromosomes in control, Wapl-MO, and Wapl-rescue oocytes at 10 hours after GVBD. Scale bar, 5 μm. (J) The rate of hyperploid eggs was recorded in control (n = 60), Wapl-MO (n = 60), and Wapl-rescue (n = 61) oocytes. Data of (B) to (D) and (J) were presented as mean percentage (mean ± SEM) of at least three independent experiments. Data of (F) and (H) were presented as mean value (mean ± SD) of at least three independent experiments. **P < 0.01, ***P < 0.001.