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PolyADP-ribosylation is required for pronuclear fusion during postfertilization in mice

PLoS ONE (2010) 5(9) 1-11
DOI: 10.1371/journal.pone.0012526
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Abstract

Background: During fertilization, pronuclear envelope breakdown (PNEB) is followed by the mingling of male and female genomes. Dynamic chromatin and protein rearrangements require posttranslational modification (PTM) for the postfertilization development. Methodology/Principal Findings:Inhibition of poly(ADP-ribose) polymerase activity (PARylation) by either PJ-34 or 5-AIQ resulted in developmental arrest of fertilized embryos at the PNEB. PARylation inhibition affects spindle bundle formation and phosphorylation of Erk molecules of metaphase II (MII) unfertilized oocytes. We found a frequent appearance of multiple pronuclei (PN) in the PARylation-inhibited embryos, suggesting defective polymerization of tubulins. Attenuated phosphorylation of lamin A/C by PARylation was detected in the PARylation-inhibited embryos at PNEB. This was associated with sustained localization of heterodomain protein 1 (HP1) at the PN of the one-cell embryos arrested by PARylation inhibition. Conclusions/Significance: Our findings indicate that PARylation is required for pronuclear fusion during postfertilization processes. These data further suggest that PARylation regulates protein dynamics essential for the beginning of mouse zygotic development. PARylation and its involving signal-pathways may represent potential targets as contraceptives. © 2010 Osada et al.

Figures

  • Figure 1. Expression of Parp, PAR level and Parg activity in the mouse oocytes. Immunofluorescence analyses of MII oocytes (A, D), embryos at 0.5 hpf (B, E) and 6 hpf (C, F) with antibody for Parp1 (A–C) and PAR (D–F). Detected antigens were colored with green. DNA was counterstained with PI, colored in red. White circles represent the outlines of pronuclei (PNs). Bars indicate 20 mm. (G) Thin layer chromatography (TLC) for the detection of poly(ADP-ribose) glycohydrolase (Parg) activity. Purified GST-Parg proteins and crude extracts from MII oocytes, parthenogenetic (activated) and untreated (IVF) embryos at 15 min and 5 hrs after activation/fertilization were loaded and reacted with synthetic PAR polymers. Release of ADP-ribose was detected by the mobility of spots from origin. A spot with high-mobility in the GST-rat Parg-loaded lane represents adenosine monophosphate (AMP). (H) RT-PCR analyses with primer sets for the 12 Parp-family genes, the Parg gene, and glyceraldehyde3-phosphatase dehydrogenase (G3pdh) gene. Amplified DNA with cDNA synthesized with reverse transcriptase (+) or without enzymes (2) was loaded in each lane. PCR reaction was carried out with the number of cycles indicated. The 100-bp ladder marker DNA was shown (M). The lowest DNA band corresponds to the 100 base pair (bp). doi:10.1371/journal.pone.0012526.g001
  • Table 1. Postfertilization development of untreated and PARP inhibitor-treated mouse embryos.
  • Figure 2. Inhibition of PNEB by PJ-34 and 5-AIQ. IVF and ICSI experiments were performed to assess the effects of Parp inhibitors at four different time-points during postfertilization development. (A) Scheme of the experimental design shows the incubation time of oocytes/embryos in normal culture medium (dotted line) or with Parp inhibitor treatment (bold line). The untreated IVF embryos reached the pronuclear stage (PN) approximately 4 hrs post-fertilization (hpf), and then underwent mitosis (Mit) from 15 to 20 hpf (open space) (B, C). Morphology of one-cell embryos at 36 hpf. Fertilized embryos were cultured for 36 hrs after insemination under untreated conditions (B) or with PJ-34 (C). A representative oocyte treated with PJ-34 shows defects in PNEB (D). DNA was counterstained with DAPI in blue. Immunofluorescence of untreated MII oocytes (MII) and PN embryos (PN), 30 mM PJ-34-treated, and 20 mM AIQ-treated PN embryos at 10 hpf with antibody for the phosphorylated form of histone H3 at serine 10 (H3 ser10, red) (E–H), and bromodeoxyuridine (BrdU, green) (M–P). DNA of embryos reacted with H3 ser10 antibody was counterstained with DAPI in blue (I–L). Frequency of the staining indicated in each panel was shown in each figure (white letters). The values in parenthesis indicate percentage of the frequency. Bars represent 25 mm. doi:10.1371/journal.pone.0012526.g002
  • Figure 3. Parp1 expression, effects of PARylation inhibition on spindle bundle formation, PARylation on tubulins. (A–D) Immunofluorescence of untreated (A, C) and MII oocytes treated with 30 mM PJ-34 for 1 hr (B, D) with antibody for Parp1 (A, B) and a/ß-tubulin (C, D). Immunosignals were colored in green. DNA was counterstained with PI (red). Frequency of the indicated images was shown in each panel (white). Values in the parenthesis indicate the percentage as the frequency (A–D). Bar represents 20 mm. (E, F). doi:10.1371/journal.pone.0012526.g003
  • Figure 4. Effect of PARylation inhibition on spindle and polynucleation during postfertilization development. Immunofluorescence with a laser-scanning confocal microscopy of diploid parthenotes (A, E) and IVF embryos at 6 (B, F), 15 (C, G) and 24 (D, H) hpf. a-Tubulin signals were colored in green with untreated (A–D) and 30 mM PJ-34-treated (E–H) embryos. White circles represent the outlines of female (Fe) and male (M) pronuclei. DNA was counterstained with PI (red). Bar represents 20 mm (H). doi:10.1371/journal.pone.0012526.g004
  • Figure 5. Biochemical analyses of PARylated proteins of MII oocytes and effects of Parp1 on the Erk-signaling. The 2D electrophoresis of 300 untreated MII oocytes with silver staining (A) and immunoblots with the monoclonal antibody for poly(ADP-ribose) polymers (clone 10H) (B). The protein spots corresponding to the immunosignals were identified as a1c (left arrowhead) and b2c (right arrowhead) tubulins by MALD-TOF mass spectrometry. (C) Western blotting of the protein extracts of MII oocytes of wild-type (WT) and Parp12/2 mice was performed with antibodies against the phosphorylated form of Erks (p-Erk 1/2), Erks, pan (Erk 1/2), Parp1, and a-tubulin as a control (C). The oocytes were collected 30 and 180 minutes after the activation of MII oocytes with Sr2+. Proteins loaded in each lane corresponded to 30 oocytes. doi:10.1371/journal.pone.0012526.g005
  • Figure 6. Defective phosphorylation of pronuclear lamins and behavior of HP1b during postfertilization development. Extracts from 100 untreated or PARylation inhibited embryos at 15 and 24 hpf were immunoblotted with antibody for phosphorylated lamin A/C, total lamin A/C, phosphorylated cdc-2, and total cdc-2 (A). Assessments for the relative phosphorylation levels of lamin and cdc2. Asterisks represent statistic significance (t test, p,0.05) (B). Immunofluorescence with laser-scanning confocal microscopy of untreated and PJ-34 treated embryos with antibodies for HP1b (C). Detected antigens were colored with green. DNA counterstained with PI was colored in red. Colocalized signals of antigens and DNA were colored in orange or yellow in merged figures. Circles (white lines) show the outlines of the female (Fe) and male (M) PNs. Other PI signals represent polar bodies (Pb). Values (percentage in parenthesis) represent frequency of the staining in each panel (C, upper panels). Bar represents 25 mm. doi:10.1371/journal.pone.0012526.g006
  • Figure 7. Scheme of spacio-temporal regulation by PARylation during perifertilization mouse development. At MII oocytes, Parp1 or other Parps are involved in spindle bundle formation mediated in part by Erk phosphorylation, which is represented as MII spindle assembly. Other Parp-associated molecules (Aurora [47]) or MAPK-associated molecules such as DOC1R [48] putatively contribute to spindle formation integrity. HP1ß loss from PNs and PN allocation are interfered by Parp inhibitors during PN stages. Phosphorylation of lamin A/C is reduced by PARylation inhibition, and subsequently PNEB is blocked. Because HP1b is associated with both chromatin and nuclear envelope, both chromatin and nuclear envelope are putatively regulated by PARylation. Duration of biological processes during 24 hrs after fertilization (open square) is listed temporally and those marked in red have been suggested to be regulated by PARylation from this study. doi:10.1371/journal.pone.0012526.g007

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APA

Osada, T., Ogino, H., Hino, T., Ichinose, S., Nakamura, K., Omori, A., … Masutani, M. (2010). PolyADP-ribosylation is required for pronuclear fusion during postfertilization in mice. PLoS ONE, 5(9), 1–11. https://doi.org/10.1371/journal.pone.0012526

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