The three-way sheet junction likely forms by loss of the membranes from one of the pronuclei and is absent in the plk-1 mutant, which fails to merge its parental genomes. (A and B) Two models to explain how three-way sheet junctions might form. (A) Removal of the membranes of the “bottom” pronucleus in the region bound by the outer–outer junctions (dashed lines) leaves behind the inner and outer membranes of the “top” pronucleus along with its holes. (B) Expansion of an outer–outer junctions (first step, gray arrows) allows juxtaposition of the inner nuclear membranes (second step, gray arrows), followed by inner nuclear membrane fusion, resulting in fenestration (final step). See text for more detail. (C) 2D cross sections from FIB-SEM image volumes (left) and segmented volumes of chromosomes (right) of pronuclei from two plk-1^ts mutant one-cell embryos grown at a semipermissive condition (23°C). Scale bars, 1 µm. (D) Reconstruction of intrapronuclear membrane structures for the same two plk-1^ts one-cell embryo at metaphase shown in D, as described in Fig. 3 F. Arrows show the approximate location of the interface. Scale bars, 1 µm. (E) Segmented subvolumes from the interface membranes of a one-cell plk-1^ts embryo at metaphase (plk-1^ts M2), viewed through the plane of the membrane interface. The membranes of the two pronuclei were labeled in red or green, as in Fig. 2 B. Scale bars, 500 nm. (F) Quantification of hole size in two pronuclear membranes at the interface of a plk-1^ts embryo at metaphase (plk-1^ts M2) compared with interface hole size in two wild-type metaphase embryos, excluding holes >1 µm². n = 252 for plk-1^ts and 159 for wild type. Note that the y axis is discontinuous, blue background highlights the upper segment of the graph (hole area >0.04 µm²). Statistical analyses were done using a two-tailed Mann-Whitney test. (G) Examples of the interface of two plk-1^ts one-cell embryos at metaphase (plk-1^ts M1 and M2). Scale bars, 1 µm. See also Video 8 (wild type) and Video 9 (plk-1^ts).