Oocyte and embryo collection
GV-stage oocytes were retrieved from the ovaries of 21–24-d-old female MF1 mice 48 h after the administration of 7 IU of pregnant male serum gonadotrophin (PMSG; Intervet) by intraperitoneal injection. Ovaries were released into warmed M2 (Sigma-Aldrich) supplemented with 200 μM IBMX (Sigma-Aldrich) to prevent GVBD and were maintained at 37°C. For oocyte maturation experiments, GV-stage oocytes were washed three times and transferred to M16 at 37°C in an atmosphere of 5% CO2
in air. To recover mature (MII) oocytes, human chorionic gonadotrophin (hCG; Intervet) was administered 48–54 h after PMSG. Oviducts were removed 14–16 h after hCG. Cumulus masses were released into M2, and cumulus cells were removed by a brief incubation in M2 containing 300 μg/ml hyalauronidase (embryo-tested grade; Sigma-Aldrich). For the recovery of pronucleate embryos, female mice were mated with males at the time of hCG administration. The embryos were recovered from the oviduct in Hepes-buffered KSOM (Lawitts and Biggers, 1993
) 27–28 h after hCG and mating. Embryos that were not immediately used were transferred to KSOM at 37°C in an atmosphere of 5% CO2
Oocytes were pressure injected using a micropipette and Narishige manipulators mounted on an inverted microscope (DM IRB; Leica). Oocytes were placed in a drop of M2 containing IBMX covered with mineral oil to prevent evaporation. Cells were immobilized with a holding pipette while the injection pipette was pushed through the zona pellucida until making contact with the oocyte plasma membrane. A brief overcompensation of negative capacitance caused the pipette tip to penetrate the cell. Microinjection was performed using a fixed pressure pulse through a picopump (World Precision Instruments). Injection volumes were estimated at 2–5% of total cell volume by cytoplasmic displacement. The oocyte volume is ~250 pl. After microinjection, the oocytes were removed in fresh drops of M2 + IBMX under oil and allowed to recover for a few minutes before any further manipulation.
Constructs and MO oligonucleotides
Human Emi1 was cloned into pCS2 + myc5 as described previously (Reimann et al., 2001a
) and pCS2-eGFP-c1 (CLONTECH Laboratories, Inc.). pCS2-GFP was a gift from M.W. Klymkowsky (Colorado University, Boulder, CO). βTrCP and βTrCPΔF were cloned in pCS2 + HA (Margottin-Goguet et al., 2003
). pMDL2–cyclin B1–GFP and pMDL2–cyclin B1Δ90
-GFP were gifts from M. Herbert (University of Newcastle, Newcastle, United Kingdom; Herbert et al., 2003
). cRNAs encoding each of these constructs were made in vitro using the mMESSAGE mMACHINE kit (Ambion). The cRNAs were polyadenylated, purified, and dissolved in nuclease-free water to a concentration of ~1 μg/μl before microinjection into GV-stage oocytes. For Emi1 knockdown in mouse oocytes, mEmi1 MO 5′-CGGGACAAGAAAGACAATGTTACTT-3′ (Gene Tools, LLC) was used at a concentration of 1.5 mM. For Cdh1 knockdown, we used an already characterized cdh1 MO (5′-CCTTCGCTCATAGTCCTGGTCCATG-3′; Reis et al., 2006
). To control for possible nonspecific effects of the MOs, a control MO was also used (5′-CCTCTTACCTCATTACAATTTATA-3′).
15 GV-stage oocytes were collected in 9 μl of nuclease-free water. After incubation with 2 U DNase I at 37°C for 30 min, the samples were subjected to oligonucleotide (dT)-primed first-strand cDNA synthesis in 20 μl by using the RETROscript kit (Ambion). The entire reaction was then used for PCR amplification with Deep Vent DNA polymerase (New England Biolabs, Inc.) for 35 cycles. The primers used for amplifying a 218-bp-long mouse Emi1 sequence were 5′-GTGGAGGTGGCAAAGACATT-3′ and 5′-GGCAAAGGACCCACTTTAC-3′. The reaction was accompanied by a negative (minus RT enzyme) control, and the experiment was repeated three times.
In situ hybridization
Ovaries of PMSG-primed mice were fixed in 4% PFA for 6 h followed by incubation in 0.5 M sucrose in PBS overnight at 4°C. The ovaries were embedded in optimal cutting temperature (Tissue-Tek), sectioned at 10 μm, and mounted on Superfrost slides (Fisher Scientific). The mouse Emi1 cDNA was subcloned into the pGEM3zf vector, linearized, and transcribed to synthesize 35S-labeled RNA probes. Hybridization mixtures with antisense and sense RNA probes were added to the slide and incubated overnight at 50°C. Post hybridization washes consisted of RNaseA treatment and decreasing concentrations of SSC washes. Hybridized slides were then dehydrated and dried. Slides were dipped into NTB2 emulsion (Kodak), exposed for 2 d, developed photographically, and counterstained with Gill's hematoxylin and eosin Y (0.5% wt/vol in ethanol). After counterstaining, tissues were cleared with xylene, mounted with Permount, visualized, and photographed with a camera (AxioCam; Carl Zeiss MicroImaging, Inc.).
3T3 cells were grown in DME supplemented with 10% FBS (Invitrogen). DMSO or 10 μM nocodazole/DMSO was added in subconfluent monolayers that were harvested 48 h later, lysed (50 mM Hepes, pH 7.5, 75 mM NaCl, 10 mM glycerophosphate, 2 mM EGTA, 15 mM MgCl2, 0.1 mM sodium orthovanadate, 1 mM DTT, 0.5% Triton X-100, and protease inhibitor cocktail [Sigma-Aldrich]), and incubated for 10 min on ice. Lysates corresponding to interphase (DMSO) or mitosis (nocodazole/DMSO) were centrifuged for 10 min at 10,000 g and 4°C. Protein concentration was determined using a protein assay kit (Bio-Rad Laboratories) according to the manufacturer's instructions. 5 μg of lysate was loaded onto gels for Western blotting.
Oocytes (200 oocytes/sample) were washed in PBS/polyvinyl alcohol (PVA) and frozen in SDS sample buffer (Laemmli, 1970
). Proteins were separated on 4–12% NuPAGE gels (Invitrogen) and transferred to polyvinylidene fluoride Immobilon-P membranes (Millipore) using the XL II Blot Module (Invitrogen). The membranes were saturated with 5% nonfat dry milk in PBS containing 0.1% Tween 20 for 1 h at room temperature and were incubated with the primary antibodies overnight at 4°C. Three antibodies were used for Western blot analysis of Emi1. 1 μg/ml rabbit polyclonal (Gentaur), a mouse monoclonal (Zymed Laboratories), and 1 μg/ml of an affinity-purified rabbit polyclonal antibody were raised against a bacterially produced myelin basic protein (MBP)−mEmi1 fusion protein. Rabbit polyclonal antibodies were affinity purified with mEmi1 fused to GST. All antibodies provide similar results. For clarity, we have presented data obtained using the Gentaur antibody. Antigen block was performed using the Gentaur antibody by preincubation for 1 h at room temperature with a threefold molar excess of human Emi1-MBP. For the detection of actin, we used 1 μg/ml of a mouse monoclonal antiactin antibody (Chemicon). Secondary IgGs conjugated to 0.05 μM HRP (goat anti–rabbit IgG or mouse IgG; Sigma-Aldrich) were incubated with the membranes for 30 min at room temperature. Immunostained bands were detected by chemiluminescence (Pierce Chemical Co.).
CDK1–cyclin B activity and MAPK activity were measured by their ability to phosphorylate histone H1 and MBP in vitro (H1 and MBP kinase assay), respectively, as previously described in detail (Marangos et al., 2003
). Eight oocytes in 2 μl M2 were transferred in 3 μl of storing solution and immediately frozen on dry ice. The samples were diluted twice by the addition of concentrated kinase buffer (Marangos et al., 2003
). The samples were then incubated at 37°C for 30 min and were analyzed by SDS-PAGE (as for Western blotting) followed by autoradiography. The autoradiographs were imaged using the phosphorimager system (Bas-100; Fuji) and analyzed with TINA 2.0 software.
A baculovirus-based expression system was used to obtain recombinant human cyclin B1–GFP at a concentration of 2 mg/ml as described previously (Marangos and Carroll, 2004a
). Sf9 insect cells infected with the baculovirus encoding His(6)−
cyclin B1–GFP were a gift from J. Pines (Gurdon Institute, University of Cambridge, Cambridge, United Kingdom).
Oocytes were fixed in freshly prepared 4% PFA (in PBS, pH 7.4, 1 mg/ml PVA) for 20 min, washed in PBS/PVA, and permeabilized in 0.1% Triton X-100 for 15 min. The cells were then incubated in blocking buffer (PBS, 3% BSA, and 10% normal goat serum) for 2 h at RT and in 5 μg/ml of a mouse anti–α-tubulin antibody (Abcam) in blocking buffer at 4°C overnight. The cells were incubated with 5 μg/ml of an AlexaFluor555-conjugated goat polyclonal anti–mouse IgG secondary antibody (Invitrogen) for 2 h at RT followed by incubation with 2 μM Hoechst 33342 to label the DNA. The immunostaining was visualized using a confocal microscope (LSM510 META; Carl Zeiss MicroImaging, Inc.). To ensure that comparisons of immunofluorescence could be made between treatment groups or developmental stages, the different samples were scanned and viewed with identical settings.
For conventional microscopy, oocytes were placed in a drop of M2 medium under oil in a chamber and placed on a microscope (Axiovert; Carl Zeiss MicroImaging, Inc.). Cyclin B1–GFP and GFP-hEmi1 were imaged using a FITC filter set (450–490-nm excitation band-pass filter, 510-nm dichroic mirror, and 505–520-nm band-pass filter for emission). Fluorescence was collected through a 20× NA 0.75 objective. For simultaneous imaging of GFP and rhodamine-dextran, the fluorophores were excited through a monochromator (Oolychrome II; Till Photonics), which was used to select excitation wavelengths of 488 and 550 nm. A triple band-pass (DAPI/FITC/TRITC) dichroic mirror was used, and emitted fluorescence was collected through a 450–490-nm band-pass filter for GFP and a 600-nm-long pass filter for rhodamine. The emitted light from all of the fluorochromes was collected using a cooled CCD camera (MicroMax; Princeton Scientific Instruments). In all imaging experiments using conventional microscopy, data were collected and analyzed using Metafluor and MetaMorph software (Universal Imaging Corp.).
A confocal microscope (LSM510; Carl Zeiss MicroImaging, Inc.) was used for the monitoring of spindles and chromosomes in the immunolocalization experiments. A 40× NA 1.3 oil immersion lens (Carl Zeiss MicroImaging, Inc.) was used. Excitation of AlexaFluor555 was provided by the 543-nm laser line of a helion-neon laser, with the laser power set at 1% of maximum. Fluorescence was collected using a 560–615-nm band-pass emission filter. For imaging Hoechst, confocal images were obtained by exciting with the 351-nm laser line of a UV laser, and emission was collected through a 435–485-nm band-pass filter. For all confocal imaging experiments, a pinhole of 2.22 Airy U was used, giving a calculated optical slice of 3.5 μm. The images were analyzed using MetaMorph software.