C. Cai et al. Xenopus (Minshall et al., 2007) and mouse (Evsikov and Marin de Evsikova, 2009a) eIF4Eb, an oocyte speciﬁc isoform of eIF4E shown to inhibit translation, has led to the devel- opment of Maskin-independent models for translational re- pression (reviewed in Standart and Minshall, 2008). In this study, we describe a novel family of KH domain proteins that, based on their expression and binding partners, are likely to participate in translational control during oocyte growth. Analysis of this gene family may help clarify how translational repression is controlled during oocyte growth in mammals. MATERIALS AND METHODS Harvesting Oocytes and Embryos All animal procedures have been approved by the Case Western Institutional Animal Care and Use Committee. CD-1 mice (Charles River Laboratories, Wilmington, MA) were used to obtain tissues, oocytes, and early embryos for RNA and protein assays. Early stage embryos (from embryonic day 0.5–5.5), fetal ovaries, and testes were obtained from naturally mated mice. To obtain germinal vesicle (GV) oocytes, the ovaries were removed from 8-wk-old mice and transferred into prewarmed M2 medium containing 100 M 3-Isobutyl- 1-methylxanthine (IBMX) (Sigma-Aldrich, St. Lous, MO), which prevents GV oocytes from undergoing germinal vesicle breakdown (GVBD). The ovaries were punctured using 27 gauge needles and GV oocytes were harvested under a stereomicroscope. To obtain GVBD and metaphase I (MI) oocytes, GV oocytes were released from IBMX and cultured for 3 and 6 h, respectively. Metaphase II (MII) arrested oocytes were recovered from mice induced to ovulate by intraperitoneal injection of pregnant mare serum gonadotropin (PMSG, Sigma-Aldrich) and human chorionic gonadotropin (hCG, Sigma- Aldrich) as described (Hogan, 1994). Ovulated oocytes were released from the ampullae of oviducts 20 h post-hCG. The cumulus cells were dispersed by brief exposure to 0.1 M hyaluronidase (Sigma-Aldrich) and careful washing. Xenopus Embryo Manipulation Xenopus embryo injections were performed as described (Iioka et al., 2009). RNA was in vitro transcribed using SP6 mMESSAGEmachine (Ambion, Aus- tin, TX). Embryos were microinjected with 5 nl per blastomere of in vitro synthesized RNA. To obtain sections of Xenopus embryos, the embryos were ﬁxed in 100% methanol then rehydrated into phosphate-buffered saline (PBS) through a methanol series (75%, 50%, 25% methanol). The embryos were cryoprotected in 15 and 30% sucrose and embedded in Tissue-Tek OCT (optimum cutting temperature) compound (Fisher Scientiﬁc, Pittsburgh, PA). Embryos were sectioned at 10 –12 m. RNA Isolation, cDNA Synthesis, and RT-PCR Heart, liver, lung, kidney, brain, intestine, muscle, testes, and ovaries were obtained from 8-wk-old mice. Ovaries and testes from fetal or young mice, oocytes, and embryos were obtained as described above. Total RNA was extracted using Trizol following the manufacturer’s protocol. After treatment with DNase I (Promega, Madison, WI), total RNA was quantiﬁed using the Quant-iT RiboGreen kit (Invitrogen). Reverse transcription was performed using the Superscript III First-Strand cDNA Synthesis kit (Invitrogen, Carls- bad, CA) in 20 l reaction. The reaction products were diluted to 200 l, and PCR was performed with primers for speciﬁc genes. Khdc1a primers: (F: CAA GGT CTG GGA AGG CTA CA; R: TGT CCC CCT TTG TTA AGT GC). Khdc1b primers: (F: GAC CAT GAG CCT TCA GCA CT; R: ACT CTG CTC AGG ACA CTG CTC). Gapdh primers: (F: AGG CCG GTG CTG AGT ATG TC; R: TCA GAT CCA CGA CGG ACA CA). Histology and RNA in Situ Hybridization In situ hybridization on ovary sections was performed as described previ- ously (Yu et al., 2008). Ovaries from eight-week-old mice were ﬁxed in 4% paraformaldehyde (PFA) for 1 h at 4°C. Ovaries were washed with PBS, cryoprotected in 30% sucrose, and embedded in OCT compound (Tissue- Tek). Tissues were sectioned at 10 m on a Leica Cryostat. Sense and anti- sense RNA probes were transcribed in vitro with the DIG labeling kit (Roche, Indianapolis, IN) from linearized T-Easy vectors (Promega). The primers for generating probes are: Khdc1a-F: CAA GGT CTG GGA AGG CTA CA; Khdc1a-R: CAG CAT TGC CTA TAC CAG AGA; Khdc1b-F: ACT TCT AGG TGT AAG ATT CTG; Khdc1b-R: AGC CCA CTG ATG TAC ACC TAC. Plasmids Used in the Study pCS2-Flag or pCS2-HA vectors were derived from the pCS2 vector (http:// sitemaker.umich.edu/dlturner.vectors/home) by adding two tandem Flag or HA tags to the original plasmids. The coding sequence of mouse Khdc1a, Khdc1b, mCpeb1, and eIf4e genes were inserted into pCS2-Flag or pCS2-HA vectors for 3138 transfections or to be linearized for in vitro transcription. For production of glutathione-S-transferas (GST)-fusion proteins, the coding sequences of mouse Khdc1a and Khdc1b genes were inserted into pGEX-4T-1 with BamHI and XhoI. pWPI, pMD2G and pCMV-dR8.74 lentivirus system (http://tronola- b.epﬂ.ch/page58115.html) was used for establishing the NIH-3T3-KHDC1 stable cell lines. The Flag-tagged Khdc1a or Khdc1b coding sequence was inserted in the pWPI plasmid using the PmeI site. Polynucleotide Binding Assay KHDC1 proteins were expressed in bacteria and puriﬁed with the B-PER GST Fusion Protein Puriﬁcation Kit (Pierce, Rockford, IL) according to the manu- facture’s protocol. The PolyU binding assay was performed as described (Chen et al., 2001) with the following modiﬁcations. Puriﬁed GST fusion protein (100 ng) and 50 l poly U agarose beads were added to 0.5 ml RNA binding buffer (10 mM Tris-HCl at pH 7.5, 100 mM NaCl, 2.5 mM MgCl2, 0.5% Triton X-100, and 0.1 mg/ml BSA). The mixture was rotated at 4°C for 30 min. The beads were spun down and washed ﬁve times with ice-cold binding buffer. Proteins bound to the beads were released by boiling in 1 SDS loading buffer and resolved on SDS-PAGE. Proteins were detected by West- ern blotting using a polyclonal antibody directed against GST (Abcam, Cam- bridge, MA). Cell Culture, Transfection, and Establishing the KHDC1 Stable Cell Lines NIH-3T3, 293T, and HeLa cells were cultured in DMEM containing 10% fetal bovine serum (FBS) at 37°C and 5% C02. Transfection was conducted using Lipofectamine 2000 according to the manufacturer’s protocol (Invitrogen). The NIH-3T3-KHDC1 stable cell lines were established as described (Wizner- owicz and Trono, 2003). Brieﬂy, 293T cells were cotransfected with pCMV- dR8.74, pMD2G, and either pWPI-KHDC1A or pWPI-KHDC1B. Twenty-four and 48 h after transfection, the medium containing lentivirus was ﬁltered and added to NIH-3T3 cells. Infected cells were passaged and analyzed by ﬂuo- rescence-activated cell sorting (FACS). GFP-positive cells were maintained as stable cells. Cell Synchronization and FACS Analysis Cells were synchronized at G2/M by thymidine-nocodazole treatment. Cells were synchronized at G1/S by double-thymidine treatment (Whitﬁeld et al., 2002). To collect cells at different stages, the cells were released from block and collected at different time points. DNA content of synchronized cells was determined by staining with 50 g/ml propidium iodide in the presence of 0.1 mg/ml RNaseA, followed by ﬂow cytometry (Coulter Epics XL, Beck- man). The data were analyzed with WinMDI software. Coimmunoprecipitation and Western Blot Analysis 293T cells were cotransfected with different plasmid combinations as neces- sary. Cells were washed with ice-cold PBS and lysed in EBC buffer: 50 mM Tris-HCl, (pH 7.5), 120 mM NaCl, 0.5% Nonidet P-40 supplemented with protease inhibitor cocktail (Roche) and PhosSTOP Phosphatase Inhibitor Cocktail (Roche). Lysates were clariﬁed by centrifugation at 16,100 g for 15 min at 4°C, and the supernatant was incubated with 2 g indicated antibody at 4°C for 2 h with rotation. Protein G agarose beads (20 l; Roche) were added for 1 h at 4°C with rotation. Then the beads were washed by EBC buffer 3 times and boiled 10 min at 95°C in 1 SDS loading buffer. For immunoblot analysis, proteins were resolved by SDS-PAGE and trans- ferred to Immobilon-P membrane (Millipore, Billerica, MA). The ﬁlters were blocked in TBST (10 mM Tris [pH 8.0], 150 mM NaCl, 0.1% Tween 20) supplemented with 5% (wt/vol) powdered milk for 1 h at room temperature and then incubated with primary antibody at room temperature for 1.5 h or 4°C overnight. Anti–pan-KHDC1rabbit serum (Rajpal et al., 2003) and afﬁnity puriﬁed KHDC1A polyclonal antibodies (GenScript, Piscataway, NJ) were used at 1:2000. Anti-GST (Abcam), anti-HA (Abcam), and anti– -tubulin (Sigma) antibodies were used at 1:10,000. The monoclonal anti-Flag antibody was used at 1:5000. The ﬁlters were then washed three times with TBST at room temperature and incubated with a horseradish peroxidase-conjugated goat anti-mouse or rabbit secondary antibody (GE, Piscataway, NJ) diluted 1:5000 in blocking buffer for 1 h at room temperature. The ﬁlters were then washed three times with TBST, and bound secondary antibody was detected by ECL plus (GE) according to the manufacturer’s protocol. Immunoﬂuorescence Staining Frozen sections of mouse ovaries and Xenopus embryos were prepared as above. The sections were rehydrated and permeabilized with 0.1% Triton X-100. Oocytes, early embryos, and cultured cells were ﬁxed with 4% para- formaldehyde (PFA) and permeabilized with 0.1% Triton X-100. Then all samples (sections, oocytes, embryos, or cells) were blocked in 3% BSA/PBS at room temperature for 1 h and incubated in diluted primary antibody at room temperature for 1.5 h or at 4°C overnight. The mAb against mCPEB1 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA) (sc-137146); KHDC1 antibodies are described above. Samples were then washed with PBS Molecular Biology of the Cell