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doi: 10.1242/10.1242/jcs.00132

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Stem-loop binding protein accumulates during oocyte maturation and is not cell-cycle-regulated in the early mouse embryo

Patrick Allard1,*, Marc J. Champigny1,*, Sarah Skoggard1,*, Judith A. Erkmann3, Michael L. Whitfield3,{ddagger}, William F. Marzluff3 and Hugh J. Clarke1,2,§

1 Departments of Obstetrics and Gynecology and Biology, McGill University, Montreal, Quebec, Canada H3A 1A1
2 Department of Medicine, McGill University, Montreal, Quebec, Canada H3A 1A1
3 Department of Biochemistry and Biophysics and Program in Molecular Biology and Biotechnology, University of North Carolina, Chapel Hill, NC 27599, USA
{ddagger} Present address: Stanford University School of Medicine, Department of Genetics, Stanford, CA 94305-5163, USA



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  		Fig. 1. Expression of SLBP in oocytes and eggs. (A) Fifty germinal vesicle (GV)-stage oocytes and metaphase II (Met II) eggs were immunoblotted using anti-SLBP antiserum (lanes 1,2) or using anti-SLBP pre-incubated with the immunogenic peptide (lanes 3,4). Molecular weight markers are indicated to the left of the blots. (B) Oocytes were stained with purified anti-SLBP and analyzed by immunofluoresence. (Upper panel) GV-stage oocyte. The arrow indicates the nucleus. (Lower panel) Metaphase II egg. The photographs were taken using identical conditions. (C) Twenty-five GV-stage (lane 2) or metaphase II (lane 3) oocytes were subjected to RT-PCR using primers derived from the mouse SLBP cDNA sequence that were expected to amplify a fragment of 800 nt. Lane 1 shows 100-nt ladder; intense band near middle of gel is 700 nt. No signal was obtained when reverse transcriptase was omitted from the reaction (not shown).

 


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  		Fig. 2. Accumulation of SLBP during oocyte maturation. GV-stage oocytes were collected and incubated for the period of time indicated at the top of each lane, then immunoblotted using the anti-SLBP antiserum. All oocytes in the 0 hour group contained a GV; all oocytes in the other groups had undergone germinal vesicle breakdown. Oocytes in the 9 hour group were separated into those that had or had not emitted the first polar body (pb). All oocytes in the 12 and 24 hour groups had produced a polar body. Lanes 1 and 2 contain 50 oocytes; all other lanes contain 25 oocytes.

 


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  		Fig. 3. (A) Abundance of SLBP during early embryogenesis. Unfertilized eggs and embryos were collected at the stages and times (hours post-hCG injection) indicated, and immunoblotted using the anti-SLBP antiserum. The inset within lanes 5-7 shows a longer exposure of the same blot. Met II, metaphase II eggs; L1c, late 1-cell; E2c, early 2-cell; L2c, late 2-cell; E4c, early 4-cell; L4c, late 4-cell. All lanes contain 25 embryos, except lane 7 which contains 100 embryos. (B) Twenty-five metaphase II oocytes (lane 1) or 1-cell (lane 2), 2-cell (lane 3), 4-cell (lane 4), 8-cell (lane 5), morula (lane 6), or blastocyst (lane 7) embryos were subjected to RT-PCR using primers derived from the mouse SLBP cDNA sequence that were expected to amplify a fragment of 800 nt. No signal was obtained when reverse transcriptase was omitted from the reaction (not shown). (C) Effect of the protein synthesis inhibitor, puromycin (PM), on SLBP quantity in early embryos. Embryos at the 1- or 2-cell stage were collected at the indicated times (hours post-hCG; lanes 1,4,7). Embryos were incubated in the presence (lanes 2,5,8) or absence (lanes 3,6,9) of puromycin for the period indicated after which embryos were analyzed by immunoblotting using the anti-SLBP antiserum. 25 embryos per lane.

 


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  		Fig. 4. Intracellular distribution of SLBP during early embryogenesis. (A) 1-cell. (B) 2-cell. (C1) Early 4-cell; DAPI stain to reveal DNA. The two lower nuclei appear to be at late telophase/early interphase. (C2) Early 4-cell; SLBP staining. (D1) 5- to 8-cell; DAPI stain. Five nuclei, one set of metaphase chromosomes and the DNA of the second polar body (arrow) are visible. (D2) 5- to 8-cell; SLBP staining. Long exposure reveals weakly stained nuclei and cytoplasm of embryo and strongly stained polar body (arrow). (Inset) Same embryo, exposure for the same duration as A-C.

 


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  		Fig. 5. (A) Stem-loop binding activity of lysates of oocytes. Lysates from 10 metaphase II oocytes (lanes 2,3) or 228 GV-stage oocytes (lanes 4,5) were incubated with a radioactive stem-loop RNA and the complexes resolved by native gel electrophoresis. In lanes 3 and 5, the antibody to SLBP was added to the extract prior to electrophoresis. (B) Stem-loop binding activity of lysates of oocytes and 1-cell embryos. Lysates from 20 oocytes or embryos were incubated with a radiolabelled stem-loop RNA and resolved on native polyacrylamide gels. MII, metaphase II oocytes; G1, 1-cell embryos, 18 hours post-hCG; S, 1-cell embryos 24 hours post-hCG; G2, 1-cell embryos 28 hours post-hCG. Presence of the SLBP antibody in the binding reaction is indicated (+). (C) Immunoblot of samples used for stem-loop binding assay. Five oocyte- or embryo-equivalents were immunoblotted using the anti-SLBP antiserum. Stages correspond to those in Fig. 5B. (D) Stem-loop binding activity of blastocysts compared with metaphase II oocytes. Lysates from equal numbers of oocytes or blastocysts were incubated with a radiolabelled stem-loop RNA and resolved on native polyacrylamide gels. MII, metaphase II oocytes; EB, early blastocysts; LB, late blastocysts.

 


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  		Fig. 6. Control of SLBP phosphorylation. (A) Extracts from 20 metaphase II oocytes were incubated without (lane 1) or with phosphatase treatment (lane 2) and immunoblotted using the anti-SLBP antiserum. (B) Dephosphorylation of SLBP following oocyte activation. Unfertilized eggs were parthenogenetically activated and samples were collected at the indicated times after activation and analyzed by immunoblotting using the anti-SLBP antiserum (lanes 2-4). Lanes 1 and 5 are oocytes arrested at metaphase II; 25 eggs per lane. (C) Control by cyclin-dependent kinase of SLBP phosphorylation. (Lanes 1-3) GV-stage oocytes were collected; one sample was harvested immediately (lane 1, 80 oocytes) and the others were incubated for 5 hours in the presence (lane 2, 75 oocytes) or absence (lane 3, 67 oocytes) of roscovitine. (Lanes 4-7) GV-stage oocytes were collected and incubated for 5 hours. Those that underwent GVBD were either harvested immediately (lane 4) or at 11 hours (lane 5) or treated for the indicated period with roscovitine (lane 6) or U0126 (lane 7); 25 oocytes per lane. (Lanes 8-10) GV-stage oocytes were collected and incubated for 12 hours. Those that produced a polar body were incubated for an additional 6 hours in control medium (lane 8) or in the presence of roscovitine (lane 9) or U0126 (lane 10); 25 oocytes per lane. All samples were immunoblotted using the anti-SLBP antiserum. The films showing lanes 1-3, 4-7, and 8-10 were not exposed for the same lengths of time. (D) Phosphorylation of SLBP at mitotic M-phase. Embryos at the 1- or 2-cell stage were collected at the indicated times (hours post-hCG). Puromycin (PM, lanes 2 and 6) or nocodazole (NCD, lanes 3 and 7) treatment was performed for the period indicated, after which embryos were collected for immunoblotting using the anti-SLBP antiserum. All control embryos had cleaved by the end of the treatment period, whereas none of the drug-treated embryos had done so; 25 eggs per lane.

 

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