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First published online 30 July 2003
doi: 10.1242/jcs.00692

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Architectural defects in pronuclei of mouse nuclear transplant embryos

Pedro N. Moreira^1,*, James M. Robl² and Philippe Collas^3,

¹ Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003, USA
² Hematech, LLC, 4401 Technology Drive, Sioux Falls, SD 57106, USA
³ Institute of Medical Biochemistry, University of Oslo, PO Box 1112 Blindern, 0317 Oslo, Norway

View larger version (52K): [in a new window]   Fig. 1. Distribution of B-type lamins, lamin A/C, NuMA and AKAP95 in mouse cumulus cells and in fertilized preimplantation embryos cultured in vitro. Immunofluorescence analysis of (A) cumulus cells and (B) mouse embryos at the pronuclear, 2-cell, 8-16-cell and blastocyst stages. (A inset) DNA. Scale bar, 10 µm. Arrow points to anti-AKAP95 labeling in the female pronucleus. (B inset) DNA labeled with Hoechst 33342. Scale bar, 50 µm. (C) MII oocytes and pronuclear stage fertilized embryos (PN) were triple-labeled using anti-AKAP95 polyclonal antibodies, anti-NuMA mAbs and Hoechst 33342 to visualize association of AKAP95 and NuMA with the female pronucleus (FPN). MPN, male pronucleus. Scale bar, 50 µm. (D) Immunoblotting analysis of cumulus cells and fertilized pronuclear stage embryos using antibodies against indicated proteins. Apparent Mr is shown in kDa.

View larger version (48K): [in a new window]   Fig. 2. Dynamics of B-type lamins, lamins A/C, NuMA and AKAP95 following NT. Disassembly of the donor nucleus and reformation of the new nucleus were monitored at the premature chromatin condensation [PCC; 3 hours post-infection (hpi)] and interphase (NT Int.; 7 hpi) stages, using antibodies against indicated proteins. Female pronuclei formed after parthenogenetic activation of MII oocytes were also analysed 6 hours after start of activation treatment (Part PN). TRITC refers to labeling with TRITC-conjugated secondary antibodies. Scale bars, 20 µm. n>20 embryos per treatment.

View larger version (36K): [in a new window]   Fig. 3. Misregulation of lamin A/C and NuMA expression in one-cell-stage NT embryos. (A) Reconstructed embryos were activated for 6 hours with SrCl2 and either no supplementation (-), 20 µg ml-1 cycloheximide (CHX) or 5 µg ml-1 actinomycin D (ActD). At the end of incubation, embryos were analysed by immunofluorescence. Anti-lamin-B (rabbit polyclonal) and anti-lamin-A/C (mAb) antibodies were used on the same preparations. Insets, DNA. Scale bar, 20 µm. (B) Relative immunofluorescence labeling intensities for indicated markers in nuclei of NT embryos treated as in (A). Reference value for each marker (100% fluorescence) represents relative amounts of immunolabeling in untreated embryos. n15 embryos per marker per treatment.

View larger version (40K): [in a new window]   Fig. 4. AKAP95 is more strongly anchored in NT pronuclei than in ICSI or parthenogenetic pronuclei. (A) Mouse one-cell-stage ICSI, parthenogenetic (Part) and NT embryos were double immunolabeled using anti-lamin-B (red) and anti-AKAP95 (green) antibodies. DNA was labeled with Hoechst 33342. Only nuclei are shown. FPN, female pronucleus; MPN, male pronucleus. Scale bars, 20 µm. (B) Mouse one-cell-stage ICSI, parthenogenetic (Part) and NT embryos, and mouse cumulus cell nuclei, were extracted with 0.1% Triton X-100, 1 mg ml-1 DNase I, 100 µg ml-1 RNase A together with 100 mM (upper two rows) or 300 mM (lower two rows) NaCl prior to fixation and double immunofluorescence analysis of lamin B (red) and AKAP95 (green). DNA is labeled blue with Hoechst 33342. Only nuclei are shown. Scale bars, 20 µm. (C) Relative proportions of unextracted lamin B, AKAP95 and DNA in nuclei of embryos or cumulus cells extracted as in (B). Fluorescence labeling intensity of each marker after in situ extraction as in (B). Reference value (100%) represents immunofluorescence labeling intensity in unextracted embryos. n10 embryos per marker per treatment.

View larger version (23K): [in a new window]   Fig. 5. Passage through first mitosis does not restore abnormalities in lamin A/C labeling and intranuclear anchoring of AKAP95 in NT embryos. (A) Two-cell stage ICSI and NT embryos were analysed by immunofluorescence using anti-lamin-A/C mAbs. (B) Two-cell-stage ICSI and NT embryos were extracted 0.1% Triton X-100, 1 mg ml-1 DNAse I, 100 µg m-1 RNase A, 300 mM NaCl prior to immunofluorescence analysis using anti-AKAP95 antibodies. DNA was labeled with propidium iodide. Scale bars, 20 µm. (C) Relative DNA and AKAP95 immunolabeling intensities in intact (100%) and extracted two-cell-stage ICSI and NT embryos. n>10 embryos per treatment.