Figure 4.

Emi2 mediates mII arrest. (A) RT–PCR for Emi1, Emi2 and β-actin transcripts and (B) Hoffman modulation microscopy, 23 h after injection of mII oocytes with EGFP, Mos, Emi1 or Emi2 siRNAs, also showing embryos 23 h after activation by exposure of mII oocytes to SrCl₂ or injection with sperm-borne oocyte activating extract (SE). Bar=50 μm. (C) Average percentages (showing ranges) of oocytes at mII 23h after siRNA injection. (D) Immunofluorescence microscopy of representative cells in (B) showing α-tubulin (green) and genomic DNA (red). Bar=20 μm. (E) Autoradiographic read-out of MBP and H1 kinase assays 22 h after injection of mII oocytes with siEmi1#2 or siEmi2#1, activation with SrCl₂ or age-matched mII oocytes that had not been treated (mII). (F) Immunoblotting of mII oocytes after injection with siRNA (+) and of aged-matched, uninjected (−) controls for Emi1 and Emi2, 22 h after injection and for Mos 10 h after injection or without injection, showing the autonomous decrease of Mos in uninjected mII oocytes. (G) Physical (left) and functional (right) analysis of baculovirus-expressed Emi2-FLAG. Arrest at mII is recorded 23 h after co-injection of siRNA with 5–15 pl freshly-prepared recombinant Emi2 protein (0.3–0.9 mg/ml) as indicated. HT, heated at 70°C for 30 min. (H) mII oocytes injected with cRNA encoding mCherry (0.15 μg/μl) or Emi2^S275N/S279N-mCherry (0.3 μg/μl) followed 10 h later by exposure to SrCl₂ and examination 9–10 h after this, showing Hoffman modulation (top panel; bar=50 μm) and percentages of oocytes apparently arrested at mII (histogram). mCherry (red) epifluorescence is shown for control embryos (mCherry) and nonpronuclear oocytes (mII) and pronuclear embryos (pn+) following injection with Emi2^S275N/S279N-mCherry cRNA. Bar=50 μm. Representative cells were subjected to MBP and H1 kinase assays (bottom right) and α-tubulin (green) and genomic DNA fluorescence microscopy as for (E and D) respectively.