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a Department of Biochemistry I, Yokohama City University School of Medicine, Yokohama 236-0004, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All experiments were carried out in strict accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction, and all protocols were approved by the Yokohama City University Committee on Animal Research. Adult female B6C3F1 mice (Sankyo Lab., Tokyo, Japan) were superovulated by i.p. injection with 5 IU of eCG (Sigma Chemical Co., St. Louis, MO) followed 48 h later by 5 IU of hCG (Sigma). The female mice were individually housed with males immediately after hCG injection and were checked for vaginal plugs the next morning as evidence for mating. All oocytes and embryos were flushed from the oviducts and uteri into sterile plastic collection dishes (35 mm in diameter) with M2 medium (Sigma) at 37°C; then morphologically well-preserved samples were selected under a microscope. Using a capillary pipette, the samples were washed 6–7 times with PBS (pH 7.4) containing 3 mg/ml polyvinylpyrrolidone (Sigma) and protease inhibitors (0.5 mM p-amidinophenylmethane-sulfonyl fluoride [Sigma]; 1 mg/ml leupeptin [Peptide Institute, Osaka, Japan]; 1 mg/ml pepstatin [Sigma]) at 37°C. One-cell embryos were obtained 28 h after hCG injection. At 12-h intervals, embryos were collected at the 2-cell, 4-cell, 8-cell, morula, and blastocyst stages. Unfertilized oocytes were collected 16 h after hCG injection from the oviducts of unmated females. Unfertilized oocytes and 1-cell embryos surrounded by cumulus cells were exposed to hyaluronidase (300 IU/ml) in M2 medium for about 5 min and then cleaned thoroughly with the same M2 medium by being repeatedly drawn in and out of a capillary pipette to shear off the cumulus cells. One to twenty oocytes or embryos in a small volume (53 nl to 1 µl) of PBS were placed at the bottom of a small well (2 x 2 mm) of a Terasaki plate (Sumitomo Bakelite, Tokyo, Japan) filled with paraffin oil to prevent evaporation, and then stored at -80°C until use. In previous work, fresh oocytes and embryos were freeze-dried, and the dry weight was individually determined by weighing on a highly sensitive quartz-fiber fish pole balance [4].
Cumulus Cell Sample
The mass of cumulus cells adhering to unfertilized oocytes was mechanically sheared off and isolated by the freehand operation of a pair of homemade glass needles in PBS, and the cell mass was used as a sample. Precise protein amounts or dry weights of the samples were not measurable, since the cell mass was too small to assay proteins and since it disintegrated on freeze-drying in attempts to prepare aggregates of freeze-dried cells. Therefore, the cumulus cell mass in about one third the volume of 20 oocytes surrounded by cumulus cells was subjected to our two-dimensional electrophoresis. This volume seemed similar to that of a group of 20 oocytes.
Human Erythrocyte Membrane Sample
As a control sample for one-dimensional microelectrophoresis and two-dimensional immunoblot, human erythrocyte ghosts were prepared using the procedure of Steck and Kant [5] and suspended at 1.43 mg of protein/ml in PBS. The protein concentration was determined by the method of Lowry et al. [6] with BSA as a standard.
One-Dimensional Microelectrophoresis
Polyacrylamide microslab gels were prepared in a small mold (37 x 37 x 1 mm; Fujiriken, Tokyo, Japan [7]), and microelectrophoresis was performed on a smaller scale than described previously [8] (see Fig. 1). A single oocyte or embryo stored frozen in a 53-nl droplet under paraffin oil was thawed, and 500 nl of lysis buffer [9] was added to the droplet. This droplet mixture contained 62.5 mM Tris-HCl (pH 6.8), 2% (w:v) SDS, 5% (v:v) mercaptoethanol, 10% (v:v) glycerol, and 0.2% (w:v) bromophenol blue as a tracking dye and was incubated at 37°C for 30 min. The mixture was added to a microwell (0.5 x 0.5 x 7 mm) of a 5% (w:v) SDS-polyacrylamide stacking gel (10 x 37 x 1 mm); electrophoresis was performed for 26 min at 5 mA to concentrate the proteins in the stacking gel and then for 22 min at 10 mA in the 10-mm range of the 10% (w:v) SDS-polyacrylamide separating gel (27 x 37 x 1 mm). The microwells were built into the stacking gel using a handmade comb, which consisted of a silicone strip (3 x 40 x 1 mm) and 13 thin silicone bars (0.5 x 0.5 x 10 mm; Disco Co., Tokyo, Japan) glued at equal spaces to the strip as teeth. After electrophoresis, the separating gel was subjected to silver staining.
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Two-Dimensional Microelectrophoresis
A separating gradient microslab gel (35 x 37 x 1 mm), consisting of a 4–17% (w:v) polyacrylamide gradient, was prepared as described above [7, 10], and microelectrophoresis was carried out based on O'Farrell's method [11]. The pH gradient ranged from 5 to 7 for the first-dimensional electrofocusing. The focusing gel solution contained 8 M urea (Gibco BRL, Gaithersberg, MD), 4% (w:v) acrylamide, 2% (w:v) 3-[(3-cholaminopropil)-dimethylamino]-1-propane sulfonate (Sigma), and 2% (v:v) ampholine pH 5–7 and 0.5% (v:v) ampholine pH 3.5–10 (Sigma). After the addition of 1.0 µl of lysis solution, the oocytes and embryos were incubated and solubilized at 37°C for 30 min in a droplet mixture containing 8 M urea, 2% (w:v) SDS, 5% (v:v) mercaptoethanol, and 2.0% (v:v) ampholines in the same ratio as for the focusing gel. The solubilized sample was loaded into a capillary tube (35 x 1 mm); isoelectric focusing was carried out for 20 min at a constant current of 0.1 mA per tube, and then for 80 min at 300 V constant voltage. For electrofocusing of proteins from a single embryo or 5 embryos, a shorter capillary (20 x 1 mm) was used to prepare a shorter gel rod to concentrate the proteins at the focusing points. After electrofocusing, the gel rod was removed from the capillary tube and placed on top of the 4–17% acrylamide gradient gel covered with equilibrium buffer containing 4.58% (w:v) Tris, 3.84 mM HCl, 0.03% (v:v) N,N,N,N-tetra-methylethylenediamine, and 2% (w:v) SDS. The acrylamide gradient spanned a microslab gel from top to bottom for electrophoresing 20 oocytes or embryos; but a half-height gradient gel (18 x 37 x 1 mm), laid on a 17% acrylamide bottom gel (17 x 37 x 1 mm), was formed for electrophoresing 1 or 5 embryos. Electrophoresis was performed at 10 mA per microslab gel for 20–45 min at room temperature. In some cases, molecular weight marker proteins were added to a well (0.5 x 0.5 x 0.5 mm) built into the left edge of the gradient gel and then electrophoresed in parallel with sample proteins electrofocused in a pH-gradient gel rod. After electrophoresis, the gradient gels were subjected to silver staining.
Silver Staining and Determination of Staining Density
After one- or two-dimensional microelectrophoresis, the resolved protein bands and spots were visualized by silver staining with a silver-staining kit (Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer's instructions. The two-dimensional patterns of the stained proteins were photographed using 400 Provia Fujichrome film (Fuji Film, Tokyo, Japan), and the images on the color slide films were scanned with a Nikon Coolscan II (Nikon, Tokyo, Japan) and stored in a Power Macintosh 8100 (Cupertino, CA). After the blue images were separated from the red and green images using Adobe Photoshop (Mountain View, CA) software, the blue-color intensities of the spots were measured using the NIH (Bethesda, MD) Image program. The gray values representing staining densities were processed using the ANOVA analysis tool of the Excel (Microsoft, Redmond, WA) program for factorial nonrepeated analysis [12].
Immunoblot Analysis after Microelectrophoresis
After two-dimensional microelectrophoresis, the separated proteins were transferred to polyvinylidene difluoride membrane (pore size, 0.45 µm; 45 x 35 mm; Millipore, Bedford, MA) at 1 mA/cm2 for 60 min. The membranes were incubated overnight at 4°C with 5% skim milk (Difco Lab., Detroit, MI) in a blocking solution containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% (v:v) Tween 20. Immunoblot analysis was carried out using antiserum against the carboxyl terminus (472-492 polypeptide, donated by Dr. Nagamatsu, S., Department of Biochemistry, School of Medicine, Kyorin University, Tokyo, Japan) of GLUT1 in the blocking solution for 2 h at room temperature. Immunoreactivity of GLUT1 was identified using a kit for streptavidin-biotin-peroxidase method (Amersham Life Science, Buckinghamshire, UK).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As a control, erythrocyte membrane proteins were electrophoresed, and clearly identifiable bands were numbered (Fig. 1) with reference to previous reports [13–15]. This signifies that our one-dimensional electrophoresis method is sound for analyzing single oocytes and embryos. According to our previous experiments, the dry weights of single oocytes and embryos show a tendency to decrease from unfertilized oocytes to blastocysts, but the average dry weight does not vary significantly among unfertilized oocytes and embryos at all preimplantation developmental stages (32.9 ± 1.9 [SD] ng dry weight for all 371 oocytes and embryos weighed; see Fig. 1 in [4]). On the plausible assumption that 50% of the dry mass is protein (see Discussion), the average protein content of single oocytes and embryos was calculated to be approximately 16.5 ng. Thus, the amounts of sample proteins were of a similar order of magnitude in erythrocyte membranes, unfertilized oocytes, and 2-cell embryos (Fig. 1). The protein bands in the oocyte in lane 4 are not clear; this indicates that the oocyte sample had degenerated. The two other oocytes showed a few bands in the range of 290 kDa (band 2, spectrin) to 72 kDa (band 4.2), including bands 3 (anion exchanger, 90 kDa) and 4.1 (glycophorin A, 79 kDa) found in erythrocyte membrane proteins [14–16]. These bands were not found or were faintly stained in the electrophoresis patterns of 2-cell embryos. In addition, broad and faintly stained bands were observable only in oocytes under the strongly stained band at approximately 70 kDa that is common to both oocytes and embryos. These findings suggest that some proteins present in unfertilized oocytes decrease in amount or disappear in 2-cell embryos (see also Fig. 2). No significant difference in the electrophoresis patterns was evident among different oocytes or different 2-cell embryos.
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Two-Dimensional Protein Patterns Observed by Silver Staining for Unfertilized Oocytes, Preimplantation Embryos, and Cumulus Cells
The overall patterns of proteins in oocytes, embryos, and cumulus cells are shown in Figure 2. Amounts of protein in 20 oocytes and embryos were approximately 330 ng, as described above. Since 30 and 60 morulae yielded visible spots similar to those of 20 morulae (data not shown), 20 oocytes and embryos were analyzed at all developmental stages. The amount of protein in the cumulus cell samples could not be determined but was thought to be similar to the amounts in 20 oocytes and embryos (for reason, see Materials and Methods). Visually the protein pattern of cumulus cells is completely different from those of unfertilized oocytes and embryos. Many more spots are observable in oocytes than in embryos, which show similar numbers of stained spots.
Surprisingly, approximately 230 spots could easily be counted in the silver-staining protein pattern of unfertilized oocytes, many of which were only faintly present or were absent from embryos. In contrast, 80–100 spots were visualized at each preimplantation stage. This signifies that the protein species expressed by maternal genes decrease or disappear after fertilization, as already suggested from the bands present in single oocytes that disappear from single 2-cell embryos (Fig. 1) in one-dimensional electrophoresis.
Staining Densities and Major Protein Spots in Preimplantation Embryos
The staining densities of BSA spots with variable areas were plotted against the amounts of BSA per spot in a model experiment (Fig. 3). The larger the amount, the stronger the density; but the relationship was not linear and seemed to saturate at high levels of density. Among all spots in embryos, eight major spots (numbered from M1 to M8 as shown in the 4-cell embryo pattern in Figure 2) were intensely stained, and those densities seemed to remain invariable during development, suggesting that the major spots represent fundamental structural or functional proteins. Similar major spots, corresponding to these 8 major spots in embryos, were detected in unfertilized oocytes, but not in the cumulus cells. The major spot distribution was different in the blastocyst as compared with 1-cell to morula-stage embryos (Fig. 2).
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The staining density curves for the 8 major spots differed significantly for three protein patterns from three different batches of 20 two-cell embryos (Fig. 4A). The average staining density of the 8 major spots was calculated as a basis for normalization. The normalized density curves did not vary significantly among batches, but the individual major spots showed significant differences (Fig. 4B). Thus, normalization is thought to be able to cancel the differences in staining efficiency for different microslab gels, retaining the characteristic staining densities of different spots. Actually, normalization produced the same effects on the major spots from different developmental stages (Fig. 4C) from 1-cell to morula, during which similarities in the appearance and position of each major spot were found (Fig. 2).
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Minor Protein Spots in Preimplantation Embryos
The other stained spots were smaller and stained more weakly than the 8 major spots; these, called minor spots, were easily localized and numbered using the major spots as a guide (Fig. 5). The distribution of minor spots was different in the blastocyst compared with other embryos from the 1-cell to the morula stage (Fig. 2). The normalized densities of these 32 minor spots in each microslab gel were plotted against the spot numbers to obtain a density curve for comparison of staining patterns among different gels (Fig. 6). The three normalized curves did not differ significantly among three gels at the 2-cell stage (Fig. 6A), showing that different staining efficiencies could be normalized as for the 8 major spots (Fig. 4). The patterns of 5 density curves for 32 spots at each stage from 1-cell to morula were significantly different (Fig. 6B), indicating that changes in protein expression patterns do occur during preimplantation development. When the 32 minor spots were divided into three groups according to the tendencies of their densities to change, 50% increased in staining density (Fig. 7A), 19% decreased (Fig. 7B), and the remainder (31%) showed variable tendencies (Fig. 7C).
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Protein Spots of One and Five Embryos
In order to elucidate differences among individual embryos, 1 and 5 one-cell embryos were analyzed in a reduced area (20 x 20 mm2) with the same gradient and pI range as for two-dimensional electrophoresis (Fig. 8). Compared with observations in the 20 embryos shown in Figures 2 and 5, fewer spots were visualized because the amount of protein in such a small number of embryos is too low for visualization of some minor spots. However, some major spots were easily detected from 1 and 5 embryos and did not appear to differ much in their relative staining densities. Nevertheless, individual differences were not obvious as far as the resolution sensitivity of the present ultra-microelectrophoretic technique is concerned.
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Two-Dimensional Micro-Immunoblot of GLUT1 in Morulae and Erythrocyte Membranes
To preliminarily demonstrate the efficiency of the present two-dimensional microelectrophoresis, the immunostaining patterns of GLUT1 are shown in Figure 9. The control erythrocyte membranes show a vaguely stained and longitudinal band centered at pI 6.6. In contrast, the morulae manifest two groups of stained spots centered at pIs 6.4 and 6.6 and distributed in a wide range of pI gradient. In each group, GLUT1 tended to self-associate in the detergent solution around the oligomeric positions of 55-kDa monomer.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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On the basis of our previous results obtained by weighing freeze-dried samples of individual mouse oocytes and embryos (Fig. 1 in [4]), we assumed that the average protein content of individual mouse oocytes and embryos is approximately 16.5 ng as 50% of dry weight or 10% of wet weight [17, 18]. Previous reports suggest average protein contents from 20 to 30 ng per oocyte or embryo based on measurement of groups of oocytes and embryos [19–21]. Thus, the amounts of protein indicated in our present report should be considered to be 50–80% of the directly measured values (Figs. 1, 5, 8, and 9). The protein content has been reported to be invariable or to have a tendency to decrease [19, 21]; we showed the same tendency for dry weight measurements in our previous report [4].
Characteristics of the One- and Two-Dimensional Microelectrophoretic Techniques
Because of the paucity of experimental materials and in order to enhance the sensitivity of analysis, many workers have applied two-dimensional electrophoresis to obtain autoradiographic patterns of protein synthesis in mouse oocytes and embryos. Samples of mouse oocytes or embryos ranging from 10 to 200 in number have been incubated with L-[35S]methionine to label the proteins for fluorography and electrophoresed using an O'Farrell [11] type two-dimensional gradient gel [22–25] or a 10% [26, 27] or 12.5% acrylamide separating gel [28]. The latter nongradient gels were used to avoid irreproducible protein patterns due to the irregular formation of the acrylamide gradient with the aim of establishing a database (e.g., QUEST system [29]). These autoradiographic studies have well characterized the protein synthesis and accumulated a wealth of data on the patterns of protein synthesis rates in mouse embryos during preimplantation development. In contrast, we miniaturized the gradient slab gels to reduce the amount of sample needed (Figs. 1, 2, 5, 8, and 9) and could obtain two-dimensional protein patterns using 20 oocytes or embryos or even a single embryo (Fig. 8), as well as one-dimensional electrophoretic patterns for single oocytes and embryos (Fig. 1). Separated proteins were silver stained because of the high sensitivity of this staining method. In preliminary experiments, the pH range 4 to 8 for electrofocusing was used (data not shown), but since more than 80% of spots were visualized in the range pH 5 to 7, this range was used for the subsequent analyses (Fig. 5). In addition, since the numbers of visible spots were similar in electrophoreses of 20, 30, and 60 two-cell embryos, 20 oocytes or embryos were analyzed (Fig. 2). Thus, we report here for the first time the actual protein patterns synthesized and present at all developmental stages of oocytes and embryos, not the patterns of protein synthesis rates as reported in previous studies. However, the number of spots visualized was limited because of arbitrary limitation of samples used for analysis (see Figs. 5 and 8).
Protein Expression Patterns of Oocytes and Embryos
Except with respect to the protein expression patterns of cumulus cells and blastocysts, the major spots remained basically similar during preimplantation development (Fig. 2). As described in Results, many minor spots disappear upon fertilization (Figs. 1 and 2), although the total amount of protein per oocyte remains almost unchanged during this period [4]. The dramatic decrease in the number of minor spots on fertilization indicates that the abundant oogenic proteins are unlikely to continue to decline for a long period in embryos after fertilization. However, the possibility should be considered that some protein spots may contain, in part, products from a few stages immediately before the analyzed stage. The distribution of major and minor spots is evidently different in blastocysts, representing the differentiation of blastomeres to the trophectoderm and inner cell mass (Fig. 2). The major and minor spot patterns of embryos from the 1-cell to the morula stage (Figs. 2 and 5) differed completely from the radiographic patterns of protein synthesis previously reported in gradient gels during the same developmental stages [23–25]. Furthermore, the large spots corresponding to our major spots were not seen in previously reported patterns of protein synthesis in nongradient gels [26–28]. Compared with the unfertilized oocyte pattern (Fig. 2), the radiographic pattern of protein synthesis in oocytes [22, 30] again differed completely from the protein spot pattern described here, and the dramatic decrease in spot number after fertilization was not detected in the pattern of protein synthesis [30]. Thus, the previous data do not appear comparable to our results.
Quantitative Analysis of Protein Spots
Although the density (gray value) of a major or minor spot is not proportional to the amount of protein in the spot, a denser spot contains a larger amount of protein (Fig. 3). Therefore, density measurement as carried out here was semiquantitative and was used for the (semi)quantitative analysis of spot density (Figs. 4, 6, and 7). Although the nature of the 8 major proteins was not clear, their densities, which remained relatively constant during preimplantation development, were successfully utilized as a basis for comparing the densities not only of major spots (Fig. 4) but also of minor spots (Figs. 6 and 7).
Although the total protein per embryo remains unchanged or shows a tendency to decrease during this period [4], the results in Figure 7 indicate that many functional proteins, which are thought to account for only a very small part of the total proteins, continue to increase in amount during preimplantation development and that their number overwhelms the number of decreasing proteins. Similarly, protein synthesis is known to increase or decrease, depending upon the autoradiographic spot, at each developmental stage [23, 25–28, 31], although the total rate of protein synthesis in developing mouse embryos has been reported to decrease from the 1-cell stage to the blastocyst stage [32].
Resolution Sensitivity and Utilization of Our Ultramicroelectrophoretic Technique
As shown in Figure 8, we tested the possibility of analyzing individual oocytes or embryos. This smaller slab gel has one fourth the area of a normal microslab gel, and 5 one-cell embryos—one fourth of the usual sample of 20 embryos—were electrophoresed (Fig. 8A). Some minor spots were too small or too faintly stained to be visualized clearly by this ultra-microelectrophoresis technique, but the major spots could be identified as in the normal microslab gel (see Figs. 2 and 5). Even a single 1-cell embryo showed clear major spots and some faintly stained minor spots. Thus, the detection capacity of silver staining seems insufficient for a single oocyte or embryo or a small number of oocytes and embryos.
Preliminary Results of Immunostaining for GLUT1 Expressed in Mouse Morulae
Application of our microelectrophoresis to GLUT1 immunoblotting was attempted, and unexpected results were obtained (Fig. 9). The blotting pattern for morulae could not be superposed on any defined silver-stained spots and the unstained area was also immunostained (see Fig. 2), since the sensitivity of immunostaining is markedly higher than that of silver staining. Similar patterns were obtained in embryos at the 2-cell and blastocyst stages (data not shown). The two-dimensional immunoblot pattern of human erythrocyte membrane ghosts has been reported to show a broad band like a smear from 45 kDa to 60 kDa with a peak at 54 kDa [33–36] and between pI 6 and 8 [34–36]. Figure 9 indicates a similar band in a similar molecular weight range and similar pI range with a peak at pI 6.6. In contrast, GLUT1 patterns of morulae consist of two groups at the pI peaks 6.6 and 6.4, and each group contains a few or more oligomeric aggregates. Two heterogenous glucose transporters, characterized later as GLUT 4 isoform ([3]), were found at pIs 6.4 and 5.6 in rat adipocytes. Both isoforms were present in low-density microsomes, and the 5.6 isoform was thought to be translocated by insulin to the plasma membranes, where only the pI 5.6 isoform was detected [37]. In addition, the erythrocyte GLUT1, although its pure form was basic (pI 8.5), is known to be electrofocused in the range of pI 6 to 7 as a result of comigration with acidic proteins (e.g., anion exchanger and other proteins) and also to have a tendency to self-associate to form protein-lipid aggregates [36, 38]. In view of these previous reports, the immunoblot patterns in Figure 9 imply the presence of two heterogeneous GLUT1 isoforms in mouse embryos and the aggregating properties of both isoforms (detailed results on GLUT1 expression to be published elsewhere). As shown here, the present microelectrophoresis techniques will be useful not only for immunoblotting, but also for analyzing the primary structures of spotted nanogram-level proteins [39] as a tool for proteome research [40, 41].
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Takahiko Kato, Department of Biochemistry I, Yokohama City University School of Medicine, Fukuura, Kanazawaku, Yokohama 236-0004, Japan. FAX: 81 45 784 4530; katomdpr{at}med.yokohama-cu.ac.jp
Accepted: January 19, 1999.
Received: September 22, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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