Fig 2: Schematic of the mechanism by which zygotes regulate the localization of paternal chromosomes. RanGTP signaling and F-actin dynamics play central roles in spatially separating paternal chromosomes from maternal chromosomes in zygotes. On the plasma membrane before fertilization, RanGTP regulates Juno/CD9 structures and blocks egg–sperm fusion in proximity to maternal chromosomes. In the cytoplasm, following egg–sperm fusion, F-actin keeps paternal chromosomes away from maternal chromosomes.

Fig 3: RanGTP activity surrounding maternal chromosomes prevents the localization of Juno and CD9. (A) Immunofluorescence images with fluorescent-labeled Juno and CD9 primary antibodies following treatment with DMSO or Noc (data of CD9 are not depicted). In Noc-treated eggs, Juno/CD9 structures are excluded around individual chromosomes. The zona pellucida was removed in A. (B) Immunofluorescence images with fluorescent-labeled Juno and CD9 primary antibodies in buffer and in RanT24N-injected eggs (data of CD9 is not depicted). In RanT24N-injected eggs, Juno/CD9 structures are formed all over the cell surface, even at regions proximal to maternal chromosomes. (C) Angle between maternal chromosomes and sperm fusion sites in buffer- or RanT24N-injected eggs. In RanT24N-injected eggs, sperm fuse at positions all over the egg surface. Data include zygotes that were polyspermic (polyspermy is 40% [buffer] and 48% [RanT24N] of the fertilized eggs). The zona pellucida was softened by treatment with glutathione. (D) Angle between maternal chromosomes and sperm fusion sites following treatment with DMSO or LatB. LatB-treated eggs exhibited significantly less spatial bias in sperm fusion compared with control eggs. However, sperm still did not fuse around maternal chromosomes. Data include zygotes that were polyspermic (polyspermy is 36% [DMSO] and 44% [LatB] of the fertilized eggs). A hole was made in the zona pellucida using a piezo-driven pipette without glutathione treatment. Fisher’s exact tests were used to obtain P values, and Holm correction was used for the correction of multiple comparison (P value of 30–60° is <8.22 × 10−10; P values of other areas are >0.05 [ns]). ***, P < 0.001.

Fig 4: Localization of Juno and CD9 defines three segments of the cell surface. (A) Immunofluorescence of Juno and CD9. Single confocal section (single slice) and maximum-intensity Z projection (Z-projection) are shown. These membrane proteins form discrete structures and exhibit polarized localization patterns. The lower panel shows high-magnification images of DA (dense Juno/CD9 area), FA (fewer Juno/CD9 area), and NA (no Juno/CD9 area). The zona pellucida was removed (A–C). (B) Average angle between the borders and maternal chromosomes. The angles from three different positions on the border for each egg were averaged. (C) Superresolution image of Juno and CD9 by Airyscan detector. There are at least two populations: one contains Juno only (circles), and the other contains both Juno and CD9. In structures containing both Juno and CD9, the intensity peaks of Juno and CD9 are not always matched (arrows).

Fig 5: F-actin contributes to but is not solely responsible for blocking the formation of Juno/CD9 structures around maternal chromosomes. (A) Immunofluorescence images of Juno and CD9 following treatment with DMSO or LatB at low magnification (whole) and high magnification (FA and DA). In LatB-treated eggs, the localization of Juno and CD9 remains overall polarized on the egg surface (whole). The FA/NA and DA/FA borders of Juno/CD9 structures become less clear in LatB-treated eggs (FA). Bleb-like long membrane protrusions containing strong Juno signals are observed in eggs treated with LatB (DA). The zona pellucida was removed (A–C). (B) Live imaging of Juno structures with a fluorescent-labeled Juno primary antibody in DMSO- or LatB-treated eggs. Juno structures in the FA region are disrupted rapidly, and Juno structures in the DA region are transferred into spot-like structures or new spot-like structures are formed de novo in the former FA region. Time-lapse video is shown in Video 5. (C) Angle between Juno/CD9 structures and maternal chromosomes after LatB treatment. The FA/DA borders in DMSO-treated eggs, and the NA/DA border in LatB-treated eggs, were determined as follows. We plotted the intensity of CD9 along a line on the maximum z-projection of image (yellow line), and the resultant plot (shown as Line plot profile) was used to acquire the position of local intensity increase (plot profile). The position was used to calculate the angle with the center of the egg in 3D. We acquired such angles from three lines per one oocyte and averaged them. For DMSO_NA/FA and LatB_closest focus, low-intensity foci of Juno/CD9 structure were manually selected (yellow and blue arrowhead). Welch’s t test was used to obtain the P value (P values between DMSO_DA and LatB_DA or LatB closest are both <0.001). ***, P < 0.001.