Ribosomal components display increased solubility in Padi6^-/- oocytes. (A) qRT-PCR analysis using 18S rRNA primers and western blot analysis using anti-S6 antibodies indicates that levels of ribosomal components are similar between Padi6^+/+ and Padi6^-/- oocytes. (B) In contrast with Padi6^+/+ oocytes, the majority of ribosomal protein S6 partitions in the supernatant of ruptured Padi6^-/- oocytes. Oocytes were ruptured in hypotonic buffer, serially centrifuged at 650 and 9000 g for 5 minutes, and the partitioning of ribosomal components into the supernatant (Sup) and pellet fractions was evaluated by immuno-slot-blot analysis using anti-S6 antibodies. (C) Transmission electron microscopic analysis indicates that although putative CPLs (arrow) are observed in the Padi6^+/+ 9000 g oocyte pellet, (D) lattices are not observed in the Padi6^-/- oocyte pellet. Arrowheads highlight ribosome-like particles associated with CPLs. Scale bar:100 nm. Values for the qRT-PCR are represented as the mean±s.e.m. from three independent experiments (P=0.407). The immunoblotting experiments were repeated three times and the electron microscopy experiments were repeated twice.

PADI6 and ribosomal protein S6 colocalize in Triton X-100 extracted oocytes and loss of PADI6 alters S6 localization but not de novo protein synthesis. (A) PADI6 (P6) colocalizes with ribosomal protein S6 in Triton X-100 extracted GV stage Padi6^+/+ oocytes and S6 localization is altered in Padi6^-/- oocytes. PADI6 and S6 co-localization in Padi6^+/+ oocytes is highlighted in merged image (M). The degree of co-localization (oval) is shown in the scatter plot. S6 localization to the oocyte cortex (or lack thereof in the Padi6^-/- oocytes) is highlighted by the arrowheads. (B) Phalloidin staining of Triton X-100 extracted GV oocytes reveals that the cortical actin network is not disrupted in Padi6^-/- oocytes. (C) Wide-field immunofluorescence analysis shows that DNMT1 displays similar cortical localization in Padi6 ^+/+ and Padi6^-/- GV stage oocytes. (D) Fluorographic analysis of [³⁵S]methionine labeled proteins from Padi6^+/+ and Padi6^-/- GV stage oocytes. Arrow indicates a protein that, based on its molecular weight (∼72 kDa) and absence from Padi6^-/-oocytes, is probably PADI6. These experiments were repeated three times.

Loss of PADI6 alters the levels and localization of ribosomal protein S6 and affects protein synthesis in two-cell embryos. (A) PADI6 (P6) colocalizes with S6 at the non-apposed cortical regions of Padi6^+/+two-cell embryo blastomeres, whereas S6 is absent from the embryonic cortex in Padi6^-/- two-cell embryos (arrows and B). (C) Phalloidin staining reveals that the cortical actin network is not disrupted in Padi6^-/- two cell embryos. (D) Western blot analysis of protein extracts indicates that S6 levels are reduced in Padi6^-/- two-cell embryos. (E) Fluorographic analysis of [³⁵S]methionine labeled proteins indicates that specific mRNAs are translated at different efficiencies in Padi6^-/- two-cell embryos. Asterisk indicates putative spindlin protein. (F) Confocal immunofluorescence analysis confirms that spindlin expression is upregulated in Padi6^-/- two-cell embryos. These experiments were repeated three times.