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Gamete Biology |
Centre de Recherche en Biologie de la Reproduction, Département des Sciences Animales, Université Laval, Sainte-Foy, Québec, Canada G1K 7P4
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gamete biology, gene regulation, oocyte development, ovum
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A cell-free incubation system was developed in the pig to allow the study of interactions between the oocyte proteins and somatic cell nuclei in a mammalian system. To identify specific proteins that remained associated with somatic cell nuclei, oocyte proteins were labeled with biotin to identify them in nuclei after coincubation. We sought to identify these proteins using proteomics techniques; however, proteomics becomes less favorable as a method for protein identification when a very limited quantity of precious material is available, such as with mammalian oocytes. The inevitable losses incurred with two-dimensional (2-D) gel electrophoresis should be avoided if the goal is to optimize use of this precious material to identify proteins of physiological importance. Using limited material, our objectives were to develop a method for identification of proteins in low abundance. The method described in this article demonstrates how 2-D gel electrophoresis can be enhanced with the use of a labeling method for proteins, their subsequent purification using immunoprecipitation, and identification using liquid chromatography-tandem mass spectrometry (LC-MSMS).
Oocyte maturation involves changes in both the nucleus and the cytoplasm, which are necessary for the oocyte to undergo fertilization and subsequent embryonic development. At this time, the oocyte synthesizes a large amount of proteins from polyadenylated RNA that has been stored during oocyte growth [7]. Protein synthesis during meiotic maturation of the porcine oocyte is essential for appropriate pronuclear development during fertilization [8], implicating these newly synthesized proteins in the process of nuclear remodeling and reprogramming. Furthermore, there is only one report of live cloned piglets using in vitro-matured oocytes [9] compared with use of oocytes matured in vivo [10, 11], suggesting that the quality of oocyte maturation and, hence, appropriate protein synthesis plays a major role in nuclear reprogramming. Also, temporal differences in protein expression exist during maturation in vitro in the pig [8] and cow [12–14]. To elucidate some of these differences, we take advantage of a cAMP analogue to block nuclear progression of the porcine oocyte during the first part of maturation in vitro in the pig, to create two distinct maturational phases. Our objectives, therefore, are to identify these newly synthesized proteins that accumulate in somatic cell nuclei with the goal to better understand the mechanisms of nuclear reprogramming. The oocyte proteins were labeled with radioactivity during two phases of maturation in vitro and those that remain associated with somatic cell nuclei after coincubation were identified using proteomics techniques and mass spectrometry.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The population of porcine ovarian epithelial-like cells used for this experiment was cells that grew inadvertently after collection and culture of porcine ovarian stromal cells. In brief, porcine ovaries were obtained from a local abattoir and transported to the laboratory in warm saline (37°C). The ovaries were dissected and follicles and granulosa cells were removed. The interstitial (stomal) tissue from the ovary was exposed by scraping the ovary with a scalpel blade, it was then minced and rinsed in sterile saline. The tissue pieces were then digested in Hanks solution supplemented with 1 mM EDTA, 0.03%(w/v) collagenase, 0.012% (w/v) DNase, and 0.006% (w/v) trypsin for 30 min in a shaking water bath at 37°C, and this digestion was repeated three times. Tissues were resuspended and allowed to pellet by gravity for 10 min. The supernatant was collected and washed in Hanks solution supplemented with 5% fetal calf serum. The cells were pelleted by centrifugation at 200 x g for 5 min, resuspended in wash medium, and recentrifuged a final time. The supernatant was removed and cells resuspended in culture medium (modified Eagle medium supplemented with 0.05 mg/ml gentamycin, 1% penicillin/streptomycin, 0.25 µg/ml amphotericin B, and 5% fetal calf serum) and seeded into culture flasks. The cell type predominant in culture showed cobblestone-like growth pattern and closely resembled ovarian surface epithelial cells. By the fourth passage, ovarian surface epithelial-like cells were the only cell type present. Cells were cultured in a humidified atmosphere of 5% CO2 at 38.4°C and passaged when they reached 70% confluency. The cells were harvested and cryopreserved at the 12th passage, after the cells had been maintained for 48 h at 100% confluency.
For cultured porcine ovarian epithelial-like cell nuclei preparation, frozen aliquots of cells (1.5 x 106 cells) were thawed, resuspended in culture medium, and then prepared according to the protocol for suspension cell lines as outlined by the NucleiPure prep nuclei isolation kit (Sigma-Aldrich, St. Louis, MO). Briefly, the cells were lysed using solution containing triton X-100, and the nuclei were purified by passage through a sucrose cushion by ultracentrifugation. Nuclei were visually checked for purity by staining with trypan blue and counted for recovery rate, which was typically between 50% and 85% nuclei of total cells at the start of the isolation procedure.
Oocyte Collection and In Vitro Maturation
Prepubertal gilt ovaries were collected at a local abattoir and transported to the laboratory in a thermoflask at 25–30°C in 0.9% saline containing antibiotics. The oocytes were then collected by aspiration of 3- to 6-mm follicles using an 18-gauge needle attached to a 10-ml disposable syringe into a 50-ml conical tube. The oocytes were allowed to sediment at room temperature for at least 10 min. The supernatant was discarded and the pellet was washed twice with Tyrode lactate (TL)-HEPES medium supplemented with 0.01% polyvinyl alcohol (PVA) [15]. The medium used for oocyte maturation was North Carolina State University (NCSU)-37 medium [16] supplemented with 10% porcine follicular fluid obtained by the puncture of follicles between 3 and 6 mm. All oocyte manipulations were performed in 0.01% PVA-TL-HEPES. Oocytes with an evenly granulated cytoplasm surrounded by compact cumulus cells were selected and were then either separated for recuperation of proteins from GV-stage oocytes or submitted to in vitro maturation (IVM) protocols. For IVM, the oocytes were washed three times in 500 µl maturation medium, which consisted of NCSU-37 medium supplemented with 00.1 mg/ml cysteine, 25 µM ß-mercaptoethanol and 10% porcine follicular fluid. Groups of 50 oocytes were placed in 500-µl of maturation medium, preincubated in four-well multidishes (Nunc, Roskilde, Denmark) overlaid with 500 µl mineral oil at 5% CO2 in a humidified atmosphere at 38.4°C. The oocytes were then incubated for the first 22 h with 10 IU/ml hCG (A.P.L., Ayerst, Montreal, Canada), 10 IU/ml eCG (Folligon, Intervet, Witby, Canada) and 1 mM of dibutyryl-cyclic AMP, then washed three times in maturation medium, and returned to another 22 h of maturation in 500 µl maturation medium without hormones and db-cAMP.
For collection of germinal vesicle (GV)-stage oocytes, the oocytes were denuded immediately after selection by vortexing for 10 min at medium speed in 100 µl of PBS (pH 7.5). For collection of metaphase II (MII)-stage oocytes, IVM oocytes were denuded in 0.01% hyaluronidase (Sigma-Aldrich) in maturation medium by vigorous pipetting. The cumulus-free GV- and MII-stage oocytes were then washed three times in PBS and frozen immediately in a minimal volume of PBS at –80°C.
Biotin Labeling of Oocyte Proteins
Aliquots of frozen GV-stage and metaphase II (MII)-stage oocytes in a minimal volume of PBS (20 µl) were thawed for biotin labeling. Proteinase inhibitors were added immediately upon thawing (0.1 mg/ml pepstatin, 1 mg/ml leupeptin, 10 mg/ml PMSF), and the oocytes were subjected to repeated freeze-thaw cycles in liquid nitrogen. Once the oocytes were lysed as determined by examination under a dissection microscope, they were centrifuged at 10 000 x g for 10 min, and the supernatant containing the oocyte proteins was removed and used for protein labeling. The oocyte proteins were labeled with 0.25 mg NHS-LC-Biotin in 12.5 µl DMSO according to the manufacturer's directions (Pierce Chemical Company, Rockford, IL), in a total reaction volume of 200 µl of PBS, pH 7.5, for 30 min at room temperature. The unreacted biotin was removed by centrifugation (5000 x g, 20 min) through a 10 000 MW cutoff ultrafiltration unit (Millipore Ultrafree-MC, Billerica, MA). The protein mixture retained on the filter was washed in 200 µl PBS three times by recentrifugation. The oocyte proteins were recovered from the filter by washing a final time with 200 µl of collecting buffer (250 mM sucrose, 50 mM KCl, 2.5 mM MgCl2-6H2O, 10 mM HEPES, 1 mM dithiothreitol, 0.01% polyvinyl alcohol) supplemented with an energy-regenerating system (ERS; 1 mM ATP, 1 mM GTP, 20 mM phosphocreatine, and 100 µg/ml creatine phosphokinase) [3], and centrifuging to a volume of 10 µl. Another 10 µl of collecting buffer with ERS was added to wash the filter, and the proteins were removed using a pipette.
Cultured ovarian epithelial-like cells were used as controls for incubation with their own nuclei, to compare protein differences and identify those proteins specifically expressed in the oocyte. The epithelial-like cell proteins were labeled following the protocol exactly as described above.
Radioactive Labeling of De Novo-Synthesized Oocyte Proteins
Groups of 50 oocytes were labeled with 0.1 mM 35S-methionine (Amersham Biosciences, Baie d'Urfé, Québec, Canada) in 500 µl maturation medium during two distinct phases of maturation. For the first maturational phase (phase A), the oocytes were labeled during the first 6 h of in vitro maturation in the presence of 10 IU/ml PMSG, 10 IU/ml hCG, and 1 mM db-cAMP to keep oocytes in GV stage. After the 6-h maturation, oocytes were washed four times in 500 µl maturation medium and returned to culture in the presence of hormones and db-cAMP for another 16 h for the first maturation, then cultured for the remaining 22 h following our standard conditions. For the second maturational phase (phase B), the oocytes were matured without radioactivity for the first 22 h of maturation under the presence of 10 IU/ml eCG and 10 IU/ml hCG and 1 mM db-cAMP to block nuclear progression. At the start of the second maturational phase (phase B), groups of 50 oocytes were incubated with 0.1 mM 35S-methionine for 6 h in maturation medium without the db-cAMP block to allow oocytes to proceed from GV stage to MII. The oocytes were washed four times in maturation medium to remove radioactivity, then replaced for a further 18 h of culture in our standard conditions. Porcine oocytes were also cultured without radioactivity to serve as a control for quality of maturation. These experiments were repeated at least three times.
After culture, all oocytes were denuded of their cumulus cells by vortexing in 100 µl PBS. The oocytes were then washed to remove cumulus cells three times in 500 µl PBS. Oocytes were then placed in a microcentrifuge tube and centrifuged for 5 min at 2000 x g. The supernatant was removed and 20 µl of collecting buffer supplemented with an energy-regenerating system containing proteinase inhibitors (0.1 mg/ml pepstatin, 1 mg/ml leupeptin, 10 mg/ml PMSF) was added. The oocytes were lysed with repeated freeze-thaw cycles and centrifuged at 10 000 x g for 10 min. The supernatant containing the oocyte proteins was then collected.
Incubation of Isolated Somatic Cell Nuclei and Labeled Oocyte Cytoplasmic Proteins
To determine which oocyte proteins become associated with somatic cell nuclei, 20 µl of labeled proteins in collecting buffer and ERS (equivalent of 150 radiolabeled oocytes or 500 biotin-labeled oocytes) were incubated with isolated ovarian epithelial-like cell nuclei (500 for radiolabeled experiment, 1000 for biotin-labeled experiment) for 1.5 h at 28°C. After incubation, the nuclei were centrifuged for 5 min at 1000 x g and the supernatant removed. They were washed two times in collecting buffer by centrifugation for 5 min at 1000 x g.
To ensure that the oocyte proteins were associated with and not attached to the surface of the nuclei, biotinylated proteins were visualized in the nuclei after incubation using immunofluorescence (procedure described below). For verification of permeability of nuclei, fluoroscein isothiocyanate (FITC)-conjugated IgG was incubated with the isolated nuclei and the nuclei were visualized using immunofluorescence.
Immunoblotting for Detection of Biotinylated Proteins
Incubated nuclei were loaded onto 10% (w/v) SDS-PAGE gels with a sample of all biotinylated proteins (equivalent of 50 oocytes). The proteins were then transferred onto nitrocellulose membranes (Hybond ECL, Amersham Biosciences). The blots were then blocked in 5% skim milk TBS-0.1% Tween-20 for 2 h, followed by incubation with 1:10 000 Neutravidin-HRP antibody (Pierce) in blocking solution for 45 min. The blots were then rinsed three times for 5 min each with TBS-Tween, detected according to the manufacturer's directions (ECL, Amersham Biosciences) and exposed to autoradiography film. Ovarian epithelial-like cell proteins served as controls for incubation with their own nuclei to compare against oocyte proteins. Each experiment was performed in triplicate.
Immunofluorescence
Nuclei were incubated on prepared poly-L-lysine (>300 000 MW) coated coverslips for 30 min at room temperature (25°C). The nuclei were then fixed and permeabilized in freshly prepared 3% paraformaldehyde in PBS with 0.3% Triton-X for 10 min. The coverslips were then washed two times in PBS for 5 min, and nonspecific binding sites on the coverslips were blocked with 5% skim milk in TBS-0.1% Tween-20 for 2 h. The coverslips were incubated with 1:100 Streptavidin conjugated with Alexa Fluor 594, and 1:100 DiOC6 (both obtained from Molecular Probes, Eugene, OR) for visualization of the nuclear membrane, in blocking solution for 30 min, washed three times for 5 min each, mounted on microscope slides in mounting medium containing Mowiol, and sealed using fingernail polish. The nuclei were visualized using a confocal microscope (Carl Zeiss Inc., Jena, Germany) equipped with neon and argon lasers.
Identification of Biotinylated Oocyte Proteins by Affinity Binding
Ovarian epithelial-like cell proteins (5.0 x 106; equivalent to 1 mg of total protein) and oocyte proteins (10 000 GV-stage oocytes; equivalent to 1 mg total protein) were biotinylated using the protocol described above except with NHS-PC-LC-Biotin (Pierce), which is photocleavable by ultraviolet (UV) light. Due to the high lipid content of the oocytes, the proteins from a second 10 000 oocyte sample were precipitated with 10% trichloric acid, 80% acetone to remove the lipids before biotinylation. The biotinylated proteins in the presence of collecting buffer with ERS were then incubated for 1.5 h with ovarian epithelial-like cell nuclei (20 000 per incubation). The nuclei were washed three times after incubation with 20 µl collecting buffer by repeated pelleting by centrifugation (1000 x g, 5 min). The nuclei were then resuspended in 200 µl PBS and sonicated using an ultrasonicator (one pulse for 5 sec, setting 4) and put immediately on ice. The biotinylated proteins recovered from the nuclei were immunoprecipitated using 0.5 ml streptavidin-linked agarose beads (Immobilized Neutravidin; Pierce) on a rotating platform for 1 h. After incubation, the beads were washed three times with 500 µl PBS each time by centrifugation (1000 x g, 5 min). The beads were then resuspended in 500 µl of 10 mM ammonium bicarbonate and transferred to a UV cuvette. The proteins were cleaved from the biotin-streptavidin bead complex using a UV transillumination box at wavelength 302 nm. The bead suspension was then transferred back to a microcentrifuge tube and the beads were pelleted by centrifugation (1000 x g, 5 min). The supernatant containing the purified proteins was removed and the beads were washed one final time with 300 µl 10 mM ammonium bicarbonate. The recuperated proteins in solution were then subjected to LC-MS/MS (Eastern Quebec Proteomics Centre, Centre Hospitalier de l'Université Laval, PQ, Canada).
Visualization of De Novo-Synthesized Proteins Using 2-D Gel Electrophoresis
Incubated washed nuclei (proteins from 150 oocytes), along with samples of total radiolabeled proteins from a total of 50 oocytes, were separated using two-dimensional SDS-PAGE techniques. The nuclei and cytoplasmic proteins were solubilized in a rehydration buffer (8 M urea, 2% Chaps, 0.5% pharmalyte, pH 3–10, 2.8 mg/ml dithiothreitol) for 1 h before loading on 13-cm Immobiline drystrips, pH 3–10 (Amersham Biosciences), overnight on an IPGphor apparatus (Amersham Biosciences) for separation of the proteins in the first dimension. The separation program included a 12-h rehydration at 30 V, 1 h at 500 V, 1 h at 1000 V, and 2 h at 8000 V. After focusing, strips were equilibrated for 15 min in 50 mM Tris-HCl, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, and bromophenol blue, and loaded onto 10% SDS-PAGE gels to separate proteins according to their molecular weight. The gels were then fixed overnight in 50% methanol, dried, and exposed to autoradiography film (Kodak BioMax) for 1 mo at –80°C for incubated nuclei or for 1 wk in Phosphoimager cassettes (Amersham Biosciences) for total radiolabeled proteins. Proteins were analyzed using ImageMaster software (Amersham Biosciences) to match spots between the gels. An average gel was made from the best two replicates, and only proteins that were common to the gels were used in the analysis. Each individual gel containing proteins remaining in incubated nuclei was cross-matched with its corresponding total protein synthesis gel to ensure that the matches using the averaged gels were accurate.
Total Oocyte Proteins for Identification
For identification of the proteins that were associated with isolated ovarian epithelial-like cell nuclei, equivalent nonradioactive gels were run using larger quantities of oocytes (4600 GV-stage oocytes and 4800 MII-stage oocytes). For each gel, due to the high lipid content of oocytes, the oocyte proteins had to be extracted using a 10% TCA, 80% acetone precipitation. In brief, oocytes were sonicated using a microtip sonicator at power setting 4 for 5 sec in 100 µl PBS containing proteinase inhibitors. Three volumes of ice-cold 10% TCA and 80% acetone were added to the lysed oocytes and incubated for 45 min at –20°C. The mixture was then centrifuged for 30 min at 10 000 x g at 4°C. The supernatant was removed, and the protein pellet was washed once with ice-cold acetone. The pellet was allowed to dry and immediately solubilized in rehydration buffer (8 M Urea, 4% Chaps, and 0.8% pharmalytes, pH 3–10, 2.8 mg/ml dithiothreitol) for 2 h at room temperature before loading the sample onto 13 cm linear, pH 3–10, Immobiline strips for the first dimension. Due to the high protein load, the focusing time was extended and a gradual increase in voltage was used. The program included a 12-h rehydration at 30 V, 100 V for 1 h, 250 V for 1 h, 500 V for 1 h, 1000 V for 1 h, 2000 V for 1 h, 4000 V for 2 h, and 8000 V, for a total of 16 000 V-h to reach a total focusing time of 28 150 V-h. After focusing, strips were equilibrated for 15 min in 50 mM Tris-HCl, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, and bromophenol blue, and loaded onto 10% SDS-PAGE gels to separate proteins according to their molecular weight. The resulting gel was then fixed overnight and proteins visualized using Bio-Safe Coomassie Blue (Bio-Rad, Hercules, CA).
Spots between the radioactive and nonradioactive gels were matched with the aid of analysis software (Imagemaster, Amersham Biosciences) and gel plugs containing the proteins of interest were excised by hand and sent for peptide mass fingerprinting (Eastern Quebec Proteomics Centre) for identification using either matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) or LC-MS/MS.
Protein Identification by MALDI-TOF and LC-MSMS
All protein identifications were performed at the Eastern Quebec Proteomics Centre. Tryptic digestions of the proteins were performed on a MassPrep liquid handling robot (Micromass, Manchester, England) according to the manufacturer's specifications and using sequencing-grade modified trypsin (Promega, Madison, WI).
Peptide tandem mass spectra (MS/MS) were obtained by capillary liquid chromatography coupled to an LCQ DecaXP (ThermoFinnigan, San Jose, CA) quadrupole ion trap mass spectrometer with a nanospray interface. An aliquot of the digested protein sample was diluted to 5 µl with 0.1% formic acid and loaded onto a reversed-phase column (PicoFrit 15 µm tip, BioBasic C18, 10 cm x 75 µm; New Objective, Woburn, MA). Peptides were eluted from the column with a linear gradient of water-acetonitrile in 0.1% formic acid at a flow rate of approximately 250 nl/ min. Mass spectra were acquired using a data-dependent acquisition mode in which each full-scan mass spectrum was followed by collision-induced dissociation of the three most intense ions. The dynamic exclusion function was enabled, and the relative collisional fragmentation energy was set to 35%. Resulting peptide MS/MS spectra were interpreted using the SEQUEST algorithm [17] and searched against proteins in the NCBI nonredundant protein database. Partial carboxamidomethylation of cysteine and oxidation of methionine were considered in the search. Confident identification of a peptide required a cross-correlation score of 1.9, 2.5, and 3.7 for singly, doubly, and triply charged peptides, respectively. Each peptide identification was confirmed by manual inspection of the spectrum.
For MALDI-TOF analysis, in-gel tryptic digestion was performed as described above. The peptides were extracted from the gel into 50% acetonitrile:water, lyophilized in a speed vacuum, and resuspended in 3µl of 0.1% trifluoroacetic acid solution. The peptide sample solution was then combined with an equal volume of matrix (
-cyano-4-hydroxycinnamic acid, 20 mg/ml in 50% acetonitrile, 0.1% trifluoroacetic acid) and spotted onto a MALDI sample plate. The sample:matrix solution was allowed to air dry at room temperature and was then washed three times with 2 µl of 0.1% trifluoroacetic acid. Mass spectra were acquired on a Voyager-DE PRO MALDI-TOF mass spectrometer (Applied Biosystems, Foster City, CA) operating in the positive-ion reflector delayed-extraction mode. Protein identifications were obtained using either ProFound (version 4.10.5, The Rockefeller University, http://prowl.rockefeller.edu/cgi-bin/ProFound) or MASCOT (http://www.MatrixScience.com) and searching the nonredundant NCBI protein database for matching peptide mass fingerprints. The search criteria used were complete carboxamidomethylation of cysteine, partial methionine oxidation, and mass deviation smaller than 60 ppm.
Immunoblotting for Detection of GRP-78
Oocyte extract (equivalent of 200 oocytes), ovarian epithelial-like cell nuclei (2000 nuclei), nuclei after incubation with oocyte proteins (equivalent of 1000 oocytes incubated with 2000 nuclei), and the supernatant from the incubation, along with a positive control for GRP 78 were loaded onto 10% (w/v) SDS-PAGE gels. The proteins were then transferred onto nitrocellulose membranes (Hybond ECL, Amersham Biosciences). The blots were then blocked in 1% skim milk TBS-0.1% Tween-20 (TBS-T) for 2 h, followed by incubation with 1:2000 GRP 78 antibody (BD Biosciences, San Jose, CA) in TBS-T for 45 min. The blots were then rinsed three times for 5 min each with TBS-T and then incubated with 1:5000 goat anti-mouse-conjugated HRP secondary antibody (Jackson Immunoresearch Laboratories, West Grove, PA) in TBS-T for 30 min. The blots were then rinsed 1 x 15 min and 3 x 5 min, and then the signal was detected according to the manufacturer's directions (ECL, Amersham Biosciences) and exposed to autoradiography film.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Nuclei were observed at the end of the incubation period, with biotinylated proteins localized inside the nuclei as shown by immunofluorescence techniques (Fig. 1). Nonincubated nuclei and nuclei incubated with unlabeled oocyte proteins were negative for staining with streptavidin (Fig. 1). The nuclei were judged as permeabilized by uptake of trypan blue dye and uptake of IgG-FITC after incubation with nuclei (data not shown).
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Biotinylation of total proteins in ovarian epithelial-like cells, GV-stage oocytes, and MII-stage oocytes resulted in immunopositive bands on the Western blots (Fig. 2). Nonbiotinylated oocyte proteins and nuclei loaded onto the gels resulted in no visible bands after immunodetection (data not shown). The proteins labeled by the biotin varied greatly between epithelial-like cells, GV-stage oocytes, and MII-stage oocytes (Fig. 2). Different bands retained within the nuclei were seen in MII-stage oocytes as compared with GV-stage oocytes, implicating synthesis of new proteins during in vitro maturation that become associated with somatic nuclei. In addition, the profiles of proteins from somatic cells differ from oocytes, showing cell-type specificity.
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A large proportion of the labeled proteins remained associated with the nuclei after incubation, regardless of cell type. This selection does not seem to simply be concentration dependent, as some proteins appear to be selectively retained by the nuclei. As shown in Figure 2, the relative proportion of an observed protein to total proteins present before incubation changes when compared with the relative amount of the protein retained by the nuclei after incubation.
Identification of Biotinylated Oocyte Proteins by Affinity Binding
The LC-MS/MS procedure resulted in four peptides that matched with known sequences in the ovarian epithelial-like cell sample (Table 1). However, in the two oocyte samples performed in this experiment, each with 10 000 oocytes, the technique was not sensitive enough to detect any peptides, even when the samples were combined for a total of proteins retained in incubated nuclei from 20 000 oocytes. The affinity-binding method did, however, improve limitations imposed by protein separation on gels, as proteins were successfully identified from the somatic cell samples that were otherwise not detectable on SDS-PAGE gels by colloidal Coomassie-blue staining (data not shown). From results obtained in our lab, we determined, by using BSA as a standard, that, if the protein spot was not visible after staining with colloidal Coomassie Blue, this was below sensitivity limits for protein identification by MALDI-TOF (data not shown).
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Detection of Radiolabeled Proteins by 2-D Gel Electrophoresis
The profiles of proteins synthesized during the first 6 h of maturation with the db-cAMP block (phase A) and the first 6 h of maturation without the db-cAMP block (phase B) are shown in Figure 3. We have visualized 208 proteins synthesized during phase A and 189 proteins synthesized during phase B based on analysis using Imagemaster software. The patterns of proteins synthesized during these two stages is very different, as only 46 of the proteins (22.1%) synthesized during phase A continued to be synthesized during phase B. The identity of several of these proteins is known, based on the sequencing of proteins from equivalent nonradioactive gels (Fig. 4 and Table 2). Nine of the identified proteins were not found to be present in GV-stage oocytes (Table 3).
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When the oocyte proteins labeled during phase A and phase B of porcine oocyte maturation were incubated with somatic cell nuclei, the oocyte proteins retained in the nuclei were detected (Fig. 3). Overall, there were 35 proteins synthesized during phase A that were found to remain associated with somatic cell nuclei, where 23 of these proteins have been identified (Tables 2 and 3). Of those proteins synthesized during the second phase of maturation, 15 proteins remain associated with somatic cell nuclei and 14 of these proteins have been identified (Tables 2 and Table 3). Comparisons across gels determined that 13 of the 35 proteins from phase A that remained associated with epithelial-like cell nuclei continued to be synthesized during the latter part of maturation.
The proteins (12 from cytoplasmic maturation and 1 from nuclear maturation) that were not identified did not have a corresponding protein present in mature oocytes. This is probably due to the sensitivity of the radioactive label-detecting proteins that are specifically synthesized during a particular stage during maturation, however, present in low abundance in oocytes.
Verification of one of these proteins, GRP-78, by Western blot analysis demonstrated that this protein is not present in nuclei before incubation with oocyte cytoplasm but enters into the nuclei after incubation (Fig. 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although it remains to be tested whether this incubation system is capable of reprogramming nuclei, there are a few factors that have been considered. As a result of labeling the oocyte proteins, the oocyte extract was activated before incubation with somatic nuclei. Activation of the oocyte results in a reduction of maturation promoting factor, which facilitates the remodeling of nuclear structure [18]. It is believed to be important to expose somatic chromatin to oocyte cytoplasm arrested in MII to allow time for the chromatin to be remodeled. However, nuclear transfer has been successful using enucleated, activated oocytes in cattle [19– 21], suggesting that the oocyte cytoplasmic proteins used in this experiment would be capable of reprogramming the nuclei. Furthermore, most frog oocyte extracts are derived from eggs or activated oocytes [22–24].
The chosen incubation temperature for the current experiment was 28°C, which seems appropriate for cell-free incubation systems. It has been suggested that nuclear import assays in mammalian cell-free extracts are to be conducted at 30°C [25]. Furthermore, another mammalian cell-free system using T cell extracts conducted incubations at 30°C, and it was shown to be capable of reprogramming nuclei [5].
The nuclei used in this experiment were permeabilized to facilitate nuclear import, as evidenced by the presence of fluorescence-labeled immunoglobin G (150 kDa) in the nuclei. Permeabilized nuclei have been used to investigate nuclear remodeling in Xenopus egg extracts [3], and it has been demonstrated that loss of the integrity of the nuclear envelope was essential for replication in terminally differentiated nuclei when exposed to Xenopus egg extracts [26]. However, use of intact nuclei has resulted in reprogramming of fibroblasts in vitro [5].
This mammalian system provides a way to observe interactions between somatic cells and the oocyte. An advantage of this system compared with nuclear-transfer procedures to elucidate these interactions is that the oocyte extract may contain important nuclear proteins that are otherwise removed by conventional enucleation procedures. A good example may be the inadvertent removal of the oocyte-specific linker histone H1oo, which localizes to chromatin upon oocyte maturation [2] or mitotic spindle components [27].
Using this cell-free incubation system, our objectives were to identify porcine oocyte proteins that remained associated with isolated cultured somatic cell nuclei after coincubation. The use of proteomics for discovery of proteins is currently limited to the more abundant proteins in a sample or to those situations where a protein can be purified in larger quantities. However, many of the proteins that are of interest in physiology may exist in very small quantities, thus demanding the development or use of sensitive techniques in proteomics for their identification. We developed a technique that allows for the use of small quantities of protein that involved labeling the oocyte proteins with biotin, enabling their identification and separation from unlabeled somatic cell nuclear proteins after coincubation by affinity binding. The purified protein mixture was directly injected into an ion trap mass spectrometer for identification of the proteins using LC-MS/MS. A similar approach has been used to reveal novel roles for cortactin by isolation of cortactin-interacting proteins [28] and to find modifications in expression of phosphorylated polypeptides related to the Scott syndrome phenotype [29]. However, with these studies, the availability of protein was not a limiting factor and immunoprecipitation techniques were combined with 2-DE for separation of proteins.
The affinity-binding procedure poses a novel way of purifying and identifying the proteins that remain associated with somatic cell nuclei during coincubation. Although we were not successful in identifying oocyte proteins, the ovarian epithelial-like cell incubation with their own nuclei resulted in a positive identification of several proteins. The lack of success with the oocyte sample should not be due to lack of protein in the initial protein mixture, as both the ovarian cell sample and oocyte sample contained 1 mg of starting material. It is possible that the high lipid content in porcine oocytes may be problematic with this method, as it may interfere with the incubation or recuperation of proteins, and further tests are warranted. This technique is also more sensitive compared with traditional protein identification methods involving proteins recovered off gels or membranes, as it limits the losses associated with gel preparation, in-gel digestion, and protein modification by staining methods. Successful identification of several proteins was achieved with this technique, even when the proteins were not abundant enough to be visible on a SDS-PAGE gel (data not shown).
Identification of the proteins from the affinity-binding technique provided interesting results and demonstrated that novel proteins and insights may be discovered by this method. We have identified one of the proteins as the cAMP-dependent protein kinase (PKA) C-alpha, which functions to phosphorylate proteins using ATP. It is present in both porcine granulosa cells [30] and porcine oocytes [31], and when activated, the catalytic subunit is translocated to the nucleus of the cell. The C-alpha isoform of PKA efficiently activates cAMP-response element (CRE)-regulated genes, and its activation compared with the C-gamma isoform may provide a means to diversify cell phenotypes [32]. The activation of the transcription factor CRE-binding protein by PKA causes the recruitment of the CREB-binding protein and p300, which are histone acetyltransferases involved in chromatin remodeling [33].
Actin was also identified using the affinity-binding technique. Actin is a very abundant cytosolic protein; however, increasing evidence suggests that it is linked to various nuclear processes ranging from chromatin remodeling to transcription to RNA splicing [34]. Actin is an integral component of a nuclear remodeling complex, BAF, and is required for the association of BAF with chromatin [4]. Furthermore, a functional mammalian nuclear reprogramming system has identified the involvement of BAF and actin in the transdifferentiation of fibroblast cells to express T cell functions [5].
Alpha-tubulin is also a protein that is purely cytoplasmic [35], and its presence in nuclei has been considered to be cytoplasmic contamination during preparation of isolated Xenopus laevis nuclei [36]. However, because the epithelial-like cell nuclei were not labeled with biotin, any protein originating from or associated with the nuclei before incubation would not have been recovered during the affinity-binding procedure; therefore, we believe that the presence of alpha-tubulin was not due to contamination. In addition, a study by Shin et al. [37] demonstrated an appearance of microtubule asters in association with decondensed chromosomes in nuclei after nuclear transfer into enucleated oocytes. This strongly suggests the presence of alpha-tubulin in nuclei incubated with cytoplasmic proteins is not an artifact or contamination.
In this study, we also sought to identify some of the de novo-synthesized oocyte proteins during in vitro maturation. Oocyte maturation involves two types of maturational stages; cytoplasmic maturation that involves reorganization of the cytoskeleton, which enables normal fertilization and further embryonic development, and also nuclear maturation, in which meiosis continues until arrest at the MII stage. One of the advantages of our in vitro system is the use of a db-cAMP block to arrest nuclear maturation during the first part of oocyte maturation in vitro, creating distinct maturational phases. The protein profiles on the 2-D gels presented two distinct patterns between the two maturational phases, as only 22% of the proteins synthesized during phase A continued to be synthesized during phase B. Similar proportions were observed during maturation in the bovine oocyte in vitro, as 16% of the proteins were synthesized throughout the 28-h in vitro maturation as analyzed by 2-D gel electrophoresis [13]. Interestingly, although Ding et al. [8] observed that inhibition of protein synthesis throughout porcine in vitro maturation impeded GVBD and pronuclear formation, protein synthesis during the period of 24–36 h of a 48-h maturation period was essential for male pronuclear formation in the pig, which roughly corresponds to our phase B. This suggests that the proteins synthesized during progression from GV stage to MII stage may play an important role in oocyte developmental competence.
The identified proteins that were associated with somatic nuclei were usually among the most abundant proteins present in GV stage- and MII-stage oocytes. This is a drawback of protein identification using proteomics, as a large quantity of protein is necessary for an identification using mass spectrometry. Interestingly, another proteomics study investigating the proteins expressed at specific stages of mouse embryo development also identified GRP 78 and HSC 70 [38], and GRP 78 was also identified on the surface of the mouse egg using proteomics [39], most likely all due to relative abundance. It is indeed possible that some of the proteins responsible for nuclear reprogramming are only present in small quantities in the oocyte and therefore would not have been chosen for identification. There were 12 proteins synthesized during phase A and 1 protein synthesized during phase B for which there were no matches present in MII oocytes, possibly due to low copy number.
This is one of the first studies to identify a large number of proteins synthesized and present in the oocyte using proteomics, demonstrating the power of this technique even in the oocyte where sample availability is a limiting factor. Currently, there are very few studies published in reproduction using proteomics techniques. Greene et al. [38] identified 42 proteins that were differentially expressed in neurulating mouse embryos. Also, mouse egg surface proteins are being characterized using proteomics in the search for new contraceptives [40]. In this study, we have identified 24 newly synthesized proteins during oocyte maturation that become associated with somatic nuclei.
Beta-actin was identified as one of the proteins associated with somatic nuclei, which confirms our findings with the affinity-binding technique. A few of the proteins identified in this study demonstrate oncogenic properties, specifically DJ-1, annexin I, and elongation factor-1 (EF-1) delta. One of the oncogenic proteins that were found to only be synthesized during the first part of maturation was identified as DJ-1. DJ-1 can transform mouse NIH3T3 cells by itself, and its expression is induced by growth stimuli [41]. It is suggested to be a novel mitogen-dependent oncogene product involved in a Ras-related signal transduction pathway [41]. Moreover, DJ-1 translocates from cytoplasm to nuclei in the S phase of the cell cycle, indicating its involvement in cell proliferation. DJ-1 has also been found to be a regulatory subunit of a 400-kDa RNA-binding protein complex [42]. Most importantly, it has been demonstrated that DJ-1 participates in fertilization [43]. It is the first report of this protein in the oocyte, and it is interesting because it is a protein implicated in fertilization and transformation. DJ-I lowers its pI upon exposure to hydroperoxide and might be a sensitive indicator of oxidative stress [44]. We have sequenced the two isoforms of the protein, and they correspond to the reported pIs of 5.8 and 6.2 for the protein, which may indicate that the oocyte was subjected to oxidative stress during in vitro maturation.
We have also identified two annexins in the oocyte, annexin I and V. They are part of a family that binds phospholipids in a calcium-dependent manner. Annexin I was not present in GV-stage oocytes and was only synthesized during phase A of maturation. Its functions include promotion of membrane fusion, exocytosis, and cortical granule release in the oocyte [45]. Annexin I is also an oncogenic protein that is involved in cell differentiation [46] and anti-inflammatory response [47]. Its expression is increased in certain carcinomas [48], and it was identified as a metastasis-associated protein in cancer cell lines by proteomics techniques [49]; however, its function in cancer is yet unknown. It is translocated into the nucleus in cancer cells [50] or by stress [51]. The presence of annexin I in the somatic nuclei after incubations may reflect a stress response or possible unknown functions.
Another oncogenic protein that has been identified is the elongation factor (EF)-1 delta. Similar to annexin I, this protein was not present in GV-stage oocytes and was only synthesized during phase A of maturation. It is a physiological substrate for cdc2 protein kinase [52] and, thus, a substrate for maturation-promoting factor, which is responsible for meiotic maturation. It serves as a phosphorylation memory signal for early development [53] and is possibly a sophisticated regulatory factor. Interestingly, the expression of EF-1 delta is upregulated in cadmium-induced carcinomas and its overexpression results in transformation and tumorigenesis [54, 55].
Annexin V, like annexin I, is a phospholipid-binding protein and has been localized to the cytoplasm in mouse oocytes [56]. Annexin V does not have the fusiogenic properties of annexin I and is involved more so in anticoagulation and ion channel activities [57]. It is a potent anticoagulant in the placenta and is essential for maintenance of placental integrity [58]. In addition, it is associated with oocyte-specific DNA methyltransferase 1 and is postulated to keep the DNMT 1o from localizing to the nucleus and thus allowing demethylation of the embryo during early cleavage stages [56].
One of the interesting proteins that we uncovered was the identification of a 20-kDa protein as the major vault protein, which is actually 100 kDa. There are three vault proteins that combine to form the 13-MDa vault complex, which is responsible for nucleo-cytoplasmic shuffling [59]. This complex is located in the cytoplasm; however, it associates with the nucleus to pick up and drop off cargo. The major vault protein is interesting, as it has also been identified as the lung-resistance protein, which is overexpressed in drug-resistant nonsmall cell lung carcinomas [60]. The 20-kDa oocyte protein was strongly matched to a splicing variant of the major vault protein [61]. The tandem mass spectrometry analysis revealed 45.7% coverage of the protein product of the gene promoter, with six matching peptides, which is abundant when we usually consider two or three peptides a good match. These results have not yet been confirmed by Western blotting; however, it is possible that the oocyte expresses a 20-kDa version of this protein.
Apolipoprotein A-1 was also found associated with somatic nuclei and was present in porcine oocytes. The oocyte contains apolipoprotein A1, as this is the high-density lipoprotein important in egg yolk, and in the chicken especially, apolipoprotein is accumulated in large quantities. This explains the presence of this protein in the oocyte; however, we are uncertain as to why it would be radioactively labeled as a synthesized protein, as the gene is specifically expressed only in the liver and intestine in mammals [62]. Interestingly, the apolipoprotein gene is unmethylated in the mature oocyte, showing the same methylation pattern as liver tissue [63], suggesting the possibility of its expression in the oocyte. It is more probable, however, that it is synthesized by cumulus granulosa cells and then transported into the oocyte, as with the chicken [64], explaining the radioactive labeling only during phase A of maturation when gap junctions between the oocyte and cumulus cells are still functional. Apolipoprotein A-1 has also been immunolocalized in the nuclei and shown to be associated with the nuclear matrix and chromatin [65].
A few of the identified proteins are stress proteins, all members of the heat shock family. These are GRP 78 or BiP protein, GRP 75/HSP 74, HSP 60, HSP 72/HSC 70. Heat shock family proteins increase their expression in response to heat stress; however, GRP 78 also responds to endoplasmic reticulum stress such as hypoglycemia [66]. These proteins aid in the correct folding of proteins, and especially after heat shock or cellular stress, they function to refold or degrade damaged proteins. HSC 70 is a cytosolic and nuclear protein, while GRP 78 is localized to the endoplasmic reticulum [67]. GRP 78 has been also reported on the surface of mouse eggs [37]. GRP 78 associates with nascent polypeptides and proteins, and its interaction appears to be related to the proteins' stability. Studies done in the Xenopus oocyte suggest that unassembled proteins remain associated with GRP78 or BiP, and it may function to stabilize these proteins during maturational arrest [68]. Interestingly, GRP 78, HSC 70, and GRP 75 are all upregulated in certain carcinomas and may be implicated in the pathogenesis of cancer [69]. Although the presence only of GRP 78 was confirmed by immunoblotting in this study, the heat shock proteins show the same pattern on a 2-D proteomic map as another study [70].
Other proteins found associated with the somatic nuclei are involved with housekeeping functions in the cell. GRP 58 is not a heat shock protein, but is also called ER 60 or thioredoxin and participates in redox reactions. ERP 28 is mainly involved with the processing of secretory proteins found associated with somatic nuclei [71]. Also, the transcription factor Lisch 7 was identified, whose expression has been shown to be upregulated by p53 in response to stress in lung cancer cells [72]. Last, cdc-42 was identified, and this kinase functions in Xenopus oocytes to polymerize actin filaments during cortical granule exocytosis [73].
In conclusion, this is one of the first studies to identify oocyte proteins that associate with somatic cell nuclei in a cell-free system using proteomics techniques. It is also a first step in building an oocyte proteomic database, as oocyte proteins are identified in association with their appearance on a 2-D proteomic map. Furthermore, the use of proteomics provides a novel way to identify oocyte proteins potentially functionally involved in nuclear reprogramming. Further studies investigating the function of these proteins in nuclear reprogramming will help in understanding the mechanisms of this phenomenon, aiding in improving nuclear transfer techniques and in vitro-fertilization procedures.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Marc-André Sirard, Département des Sciences Animales, Pavillon Paul-Comtois, Université Laval, Sainte-Foy, PQ, Canada G1K 7P4. FAX: 418 656 3766; marc-andre.sirard{at}crbr.ulaval.ca
Received: 6 January 2004.
First decision: 20 February 2004.
Accepted: 4 May 2004.
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