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First published online 3 July 2008
doi: 10.1242/dev.016329

Development 135, 2627-2636 (2008)
Published by The Company of Biologists 2008
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Role for PADI6 and the cytoplasmic lattices in ribosomal storage in oocytes and translational control in the early mouse embryo

Piraye Yurttas1,2,*, Alejandra M. Vitale1,*, Robert J. Fitzhenry1, Leona Cohen-Gould3, Wenzhu Wu1, Jan A. Gossen4 and Scott A. Coonrod1,{dagger},{ddagger}

1 Department of Genetic Medicine, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY, USA.
2 Weill Graduate School of Medical Sciences of Cornell University, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY, USA.
3 Department of Cell and Developmental Biology, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA.
4 NV Organon, Oss, The Netherlands.


Figure 1
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  		Fig. 1. PADI6 is required for cytoplasmic lattice formation in oocytes and two-cell embryos. (A) Padi6+/+ primordial follicle oocyte. N, nucleus; C, cytoplasm. (B) Padi6-/- primordial follicle oocyte. (C) CPLs are first observed in Padi6+/+ growing oocytes. Rb, ribosome; IS, intermediate structure; CPL, cytoplasmic lattice. (D) CPLs do not form in Padi6-/- growing oocytes. (E) CPLs are also observed in Padi6+/+ fully grown germinal vesicle stage oocytes. (F) CPLs are not observed in Padi6-/- fully grown germinal vesicle stage oocytes. (G) Arrows highlight CPLs in Padi6+/+ two-cell embryos. (H) CPLs are not observed in Padi6-/- two-cell embryos. Scale bar: 200 nm. These experiments were repeated twice.

 

Figure 2
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  		Fig. 2. 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.

 

Figure 3
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  		Fig. 3. 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.

 

Figure 4
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  		Fig. 4. 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.

 

Figure 5
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  		Fig. 5. Embryonic genome activation is defective in Padi6-/- two-cell embryos. (A) Levels of RNA polymerase II (RNA Pol II), phosphorylated RNA Pol II (P-Ser2-CTD) and acetylated histone H4 (H4K5 acetyl) are significantly reduced in Padi6-/- two-cell embryos. Representative nuclei shown were chosen based on their average signal intensity matching the respective mean of average intensities for Padi6+/+ and Padi6-/- embryo nuclei. Values represent the mean signal intensity±s.e.m. (B) BrUTP incorporation into the nucleus of Padi6-/- two-cell embryos is significantly reduced. Representative nuclei are shown above bars in histogram. Values represent the mean BrUTP nuclear incorporation±s.e.m. from three independent experiments (P<0.05). (C) Fluorographic analysis of [35S]methionine labeled and Triton X-100 extracted two-cell embryos indicates that synthesis of transcription requiring complex (TRC) proteins is reduced in Padi6-/- embryos. These experiments were repeated three times.

 

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