Involvement of Histone H3 (Ser10) Phosphorylation in Chromosome: MATERIALS AND METHODS
Culture of Pig Oocytes
Pig ovaries were obtained from prepubertal gilts at a local slaughterhouse. The ovaries were washed once with 0.2% (w/v) cetyltrimethylam-monium bromide and twice with Dulbecco phosphate-buffered saline (PBS) containing 0.1% (w/v) polyvinyl alcohol (PbS-PVA; Sigma Chemical Co., St. Louis, MO). Antral follicles 4-6 mm in diameter were dissected in PBS-PVA from ovaries following the technique described by Moor and Trounson. After being opened in 25 mM HEPES buffered TCM-199 (Earl salt; Nissui Pharmaceutical, Tokyo, Japan), oocytes-cu-mulus complexes with a piece of parietal granulosa tissue (oocyte-cumu-lus-granulosa cell complexes; OCGCs) were isolated from the follicles. Following two washes in HEPES buffered TCM-199, the OCGCs were cultured in 2 ml of maturation medium. The maturation medium was bicarbonate-buffered TCM-199 supplemented with 10% (v/v) heat-treated fetal calf serum (Biocell Inc., Carson, CA), 0.1 mg/ml sodium pyruvate, 0.08 mg/ml kanamycin sulphate (Sigma Chemical Co.), 2.2 mg/ml sodium bicarbonate, and 0.1 IU/ml human menopausal gonadotropin (Pergonal; Teikoku Zoki, Tokyo, Japan), and two everted theca shells with gentle agitation in an atmosphere of 5% CO2 in humidified air at 38.5°C. The OCGCs were cultured for various times to obtain oocytes at the germinal vesicle (0 h), diakinesis (18 h), metaphase I (24-27 h), anaphase I and telophase I (30-33 h), and metaphase II (42 h) stages.
To examine the effects of the inhibition of PP1/PP2A on chromosome condensation, oocytes-cumulus complexes (OCCs) were used. The OCCs were cultured in maturation medium containing 50 nM calyculin A (CL-A; Sigma-Aldrich, Germany) or 2.5 |xM okadaic acid (OA; sodium salt; Sigma-Aldrich) for 0, 0.5, 1, 2, 3, 4, and 6 h. To inhibit the activation of MAP kinase during the PP1/PP2A inhibition, the OCCs were cultured in maturation medium containing 50 nM CL-A and an MEK inhibitor, 0.1 mM U0126 (Promega, Madison, WI).
After culturing, the oocytes were denuded completely by pipetting and used for immunostaining or kinase activity assay.
After being washed twice in PBS-PVA, the denuded oocytes were fixed in PBS-PVA containing 4% (w/v) paraformaldehyde and 0.2% (v/v) Triton X-100 for 40 min. The fixed oocytes were washed twice in PBS-PVA for 15 min each and stored overnight in 1% (w/v) bovine serum albumin (BSA; International Regents Corporation, Kobe, Japan) supplemented with PBS-PVA (BSA-PBS-PVA) at 4°C. The oocytes were blocked with 10% (v/v) goat serum (DakoCytomation A/S, Glostrup, Denmark) in BSA-PBS-PVA for 45 min and then incubated for double labeling in a mixture of rabbit polyclonal anti-phospho-histone H3 at Ser10 (1: 100 dilution; #9701; Cell Signaling Technology Inc., Beverly, MA) and mouse monoclonal anti-lamin A/C (1:100 dilution; sc-7292, Santa Cruz Biotechnology Inc., Santa Cruz, CA) antibodies at 4°C overnight. After being washed three times in BSA-PBS-PVA for 15 min each, the oocytes were incubated in the mixture of Alexa Fluor 488 labeled goat-anti rabbit IgG (1:400 dilution; Molecular Probes Inc., Eugene, OR) and Alexa Fluor 350-labeled goat-anti mouse IgG (1:400 dilution; Molecular Probes Inc.) as the conjugated second antibodies for 40 min at room temperature. After being washed three times in BSA-PBS-PVA for 15 min each, the chromosomes were stained with propidium iodide (400 |xg/ml; Sigma Chemical Co.). Following complete washing, the oocytes were mounted on slides by Vectashield mounting medium (Vector Laboratories Inc., Burlingame, CA) and observed under a fluorescent microscope (U-LH100HGAPO; Olympus Optical Co., Tokyo, Japan). Determination of the meiotic stages was based on the chromosome configuration described by Motlik and Fulka. In the negative control, the oocytes were reacted with nonimmune rabbit serum instead of rabbit polyclonal anti-phospho-histone H3 at Ser10 antibody or with mouse IgG instead of mouse monoclonal anti-lamin A/C antibody.
After denudation and three washes in PBS-PVA, each group of oocytes was transferred into an Eppendorf tube with 1 |xl of PBS-PVA for double kinase assay following the technique described by Kanayama et al.. Each sample contained two oocytes for double assay of Cdc2 kinase and MAP kinase and five oocytes for double assay of Cdc2 kinase and histone H3 kinase. Thereafter, 4 |xl of ice cold extraction buffer was added, and the samples were frozen at —80°C before the assay. The extraction buffer was composed of 80 mM p-glycerophosphate, 25 mM HEPES (pH 7.2), 20 mM EGTA, 15 mM MgCl2, 1 mM DTT, 1 mM APMSF, 0.1 mM Na3VO4, 1 |xg/ml leupeptin (Sigma Chemical Co.) and 1 |xg/ml aprotinin (Sigma Chemical Co.). To examine the kinase activities during maturation, the meiotic stage of the oocytes was determined before they were transferred into the Eppendorf tubes by staining with 12 |xg/ml Hoechst 33342 (Polysciences Inc., Warrington, PA) for 20 min followed by observation under a fluorescent microscope. After being thawed, the oocytes were centrifuged at 13 000 X g for 10 min, added with 5 |xl of kinase buffer and 5 |xl of substrate solution, and incubated for 20 min at 37°C. The kinase buffer was composed of 75 mM HEPES (pH 7.2), 75 mM p-glycerophosphate, 75 mM MgCl2, 6 mM DTT, 10 mM EGTA, 60 |xM ATP, 15 |xM cAMP-dependent protein kinase inhibitor peptide (Sigma Chemical Co.) and 0.3 jLxCi/^l [^-32P]ATP (250 ^Ci/25 |xl; Amersham Pharmacia Biotech, Buckinghamshire, UK). The substrate solution for the double assay of the Cdc2 kinase and MAP kinase was composed of 4.25 |xl of histone H1 (5 mg/ml, from calf thymus; Boehringer, Tokyo, Japan) and 0.75 |xl of myelin basic protein (5 mg/ml, MBP from bovine brain; Sigma Chemical Co.). The substrate solution for the double assay of Cdc2 kinase and histone H3 kinase was composed of 2.5 |xl of histone H1 and 2.5 |xl of histone H3 (1 mg/ml, from calf thymus; Boehringer). The reaction was terminated by the addition of 5 |xl of 4X SDS sample buffer and boiling for 5 min. The samples were loaded onto a 15% gel for separation of labeled myelin basic protein and histone H1, or histone H3 and histone H1. After running, the gels were dried and autoradiographed. The autoradiograms were scanned with an image analysis system with Image Master ID Elite software Version 3.00 (Amersham Pharmacia Biotech). The kinase activity in the oocytes at metaphase I was arbitrarily set to 100%, and the other bands were expressed relative to that as a mean percentage ± SEM.
At least 30 immunostained oocytes were examined by immunofluores-cent microscopy in each group. Each kinase assay experiment was performed in at least three replicates. Statistical differences in the activities of the kinases were analyzed by one-way analysis of variance (F1-test) followed by the Tukey multiple range test. Other values were analyzed using the chi-square test. P values less than 0.05 were considered statistically significant.