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Gamete Biology |
Institute of Animal Physiology and Genetics,3 Academy of Sciences of the Czech Republic, 277 21 Libechov, Czech Republic
Institute of Microbiology,4 Academy of Sciences of the Czech Republic, 142 20 Prague, Czech Republic
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gamete biology, gene regulation, in vitro fertilization, in vitro maturation, mass spectrometry, meiosis, oocyte development, pig oocyte, proteome, two-dimensional gel electrophoresis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In spite of this tendency, our knowledge of molecular networks underlying fine mechanisms of reproductive processes remains elusive. In contrast with this field, various omics, such as genomics, transcriptomics, proteomics, and metabolomics, represent strategies that are currently mostly used to improve understanding of many other biological processes at a molecular level. Huge amounts of information on gene expression, cellular proteins, or whole tissues are organized in databases, and such resources, together with in silico approaches, offer only a starting point for a more comprehensive understanding of cellular and tissues functions in health and disease.
To date, there have been relatively few studies examining genomes and proteomes of whole tissues important for reproductive function, germ cells as well as embryos [1– 5]. Molecular investigations of gene expression or protein composition of germ cells or embryos have been sparse due to the paucity of sample cells and sufficiently sensitive procedures to analyze and identify them. With the progress of technologies, including linear amplification of cDNA populations, it has been possible to consider gene profiling in this biological model of extremely limited availability [6– 9]. Recently, it was suggested that integrated projects involving specialists in embryology, reproductive biotechnology, genomics, and proteomics are necessary to gain a holistic view of fertilization and intact embryo-maternal communication [10, 11]. Deeper knowledge of these mechanisms should help improve poor developmental potential of in vitro-produced embryos, their successful implantation, and maintenance of pregnancy.
The mammalian oocyte is the cornerstone of reproductive biology. When fully grown oocytes are removed from their follicles, they can resume meiosis and mature spontaneously under in vitro conditions. However, nuclear maturation under in vitro conditions is not accompanied by complete cytoplasmic maturation, which is essential for successful fertilization and the initiation of zygotic development [12–14]. Nevertheless, it has been proved that transcripts and proteins synthesized and stored earlier, during the period of oocyte growth and completion of all these metabolic steps, allows acquisition of a full meiotic and developmental competence [15]. Transcription activity of the oocyte rapidly decreases during maturation; therefore, it is expected that the important information necessary for full meiotic and developmental competence of oocytes be retained at the level of proteins. However, the molecular events responsible for these processes are not known. In this study, we have used a proteomics approach to analyze protein patterns of pig oocytes during in vitro maturation. Furthermore, we initiated comparative analysis of the oocytes at different stages of maturation to characterize candidate proteins that are differentially expressed during in vitro maturation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tissue culture medium 199 (TCM 199) was from Sevac (Prague, Czech Republic), fetal calf serum was provided by Bioveta (Ivanovice, Czech Republic), and porcine follicle-stimulating hormone was from Biogenesis (Poole, UK). Immobiline Dry Strips (IPG), pH 3–10 NL, 7 cm; and ampholytes, pH 3–10, were purchased from Amersham Pharmacia Biotech (Uppsala, Sweden); 3-([3-cholamidopropyl] dimethylammonio)-2-hydroxyl-1-propanesulfonate (CHAPS), urea, and dithiothreitol (DTT) were from Amersham Pharmacia Biotech; acrylamide/bis-acrylamide 30% solution, Tris-HCl, agarose, iodoacetamide, thiourea, glycine, silver nitrate, protease inhibitor complete tablets, and trifluoroacetic acid (TFA) were from Sigma (St. Louis, MO); ammonium persulfate, sodium dodecyl sulphate (SDS), and N'N'N''N''-tetramethylenediamine were from Bio-Rad (Richmond, CA); tributyl phosphine was purchased from Fluka (Buchs, Switzerland).
Oocyte Collection and Culture
Ovaries, collected from slaughtered pigs, were transported in physiological saline at 20°C to the laboratory. The ovaries were briefly washed for 20 sec in 70% ethanol and then twice in physiological saline. The oocytes were obtained by aspiration of antral follicles about 5 mm in diameter. Only oocytes surrounded by compact cumuli were used for culture. Oocytes were cultured in droplets of TCM 199 medium supplemented with 10% fetal calf serum, 100 ng porcine follicle-stimulating hormone, and antibiotics at 38°C in an atmosphere of 5% CO2. The samples were collected at 0 (GV), 28 (MI), and 45 (MII) h during spontaneous in vitro oocyte maturation. At the end of culture, the cumulus and corona radiata of the oocytes were removed by mechanical stripping of cumulus cells with a manipulation pipette. Denuded oocytes were then washed by physiological saline and after the last wash, the oocytes were stored at –80°C until use in the experiment. Morphological evaluation of oocytes was used to verify GV, MI, or MII stage of in vitro maturation and quality of the oocytes collected for two-dimensional gel electrophoresis (2-DE). The oocytes were mounted on microscope slides with vaseline, covered with a cover glass, and fixed in ethanol:acetic acid 3:1 for 24 h. Staining was performed with 2% orcein in 50% aqueous-acetic acid, 1% sodium citrate. The slides were then placed in 40% acetic acid and observed with a phase-contrast NU Zeiss microscope (Jena, Germany). The collection of the oocytes used for the proteomic study was based on the criteria that at least 98% of oocytes reached an appropriate maturation stage.
Sample Preparation and Two-Dimensional Gel Electrophoresis
For analytical 2-DE, samples of 200 oocytes were lysed in 30 µl of lysis buffer containing urea (9 M), CHAPS (4% w/v), Tris (40 mM), DTT (70 mM), 2% (v/v), ampholytes (pH 3–10), protease inhibitors, and a trace of bromophenol blue. The samples were loaded by gel rehydration on 7-cm immobilized, pH 3–10, nonlinear gradient strips for 2-DE. The separations were performed as described by Hochstrasser et al. [16]. The isoelectric focusing was carried out in a Multiphore II apparatus with a 3500-V power supply. The second dimension was done in 12% polyacrylamide gels using a MiniProtean II cell. A sensitive ammoniacal silver staining visualized protein spots [17].
Silver-stained gels were scanned using a laser densitometer (Duo Scan, AGFA, 2088 x 1872 pixels, 16 bits/pixel) generating 7.5-Mb images. The images were evaluated by PD Quest analysis software version 7.1 (Bio-Rad). For each gel, the spots were detected and quantified automatically using default spot detection. A manual spot editing was performed and the results were in agreement with those of the visual inspection. Quantification of spots was done in terms of parts per million. To compare and analyze the images of the gels in experiments, the MatchSet Tool was used and the Master, a synthetic image containing the data from all the gels in the MatchSet was created. Three independently prepared samples for each stage of oocyte maturation were evaluated. The Analysis Set Manager, including Student t-test, was used for determination of significant protein spot differences at the level of P < 0.05.
Enzymatic In-Gel Digestion
For the micropreparative 2-DE, up to 600 oocytes were loaded on IPG strips. The spots stained by Coomassie brilliant blue G250 (CBB) were excised from the gel, cut into small pieces, and washed several times with 10 mM dithiotreitol, 0.1 M 4-ethylmorpholine acetate (pH 8.1) in 50% acetonitrile (MeCN). After complete destaining, the gel was washed with water, shrunk by dehydration in MeCN, and reswelled again in water. The supernatant was removed and the gel was partly dried in a SpeedVac concentrator. The gel pieces were then reconstituted in a cleavage buffer containing 0.01% 2-mercaptoethanol, 0.1 M 4-ethylmorpholine acetate, 1 mM CaCl2, 10% MeCN, and sequencing-grade trypsin (50 ng/µl; Promega, Madison, WI). After overnight digestion, the resulting peptides were extracted to 40% MeCN/0.5% TFA. The samples were purified and concentrated using C18 ZipTips (Millipore, Bedford, MA) before mass spectrometric analysis.
MALDI Mass Spectrometry
A saturated solution of 
-cyano-4-hydroxycinnamic acid (Sigma, Steinheim, Germany) in aqueous 50% MeCN/0.2%TFA was used as a MALDI matrix. A 2-µl sample and 2 µl of matrix solution were premixed in a tube, and 0.5 µl of the mixture was placed on the sample target and allowed to dry at ambient temperature. Positive ion MALDI mass spectra were measured on a Bruker BIFLEX II reflectron time-of-flight mass spectrometer (Bruker-Franzen, Bremen, Germany) equipped with a SCOUT 26 sample inlet, a gridless delayed extraction ion source, and a nitrogen laser (337 nm; Laser Science, Cambridge, MA). Ion acceleration voltage was 19 kV and the reflectron voltage was set to 20 kV. The spectrometer was calibrated externally using the monoisotopic [M+H]+ ions of peptide standards angiotensin II and insulin (Sigma). Proteins were identified by searching of peptide mass maps in Swiss-Prot or NCBInr database using the search program ProFound (http://129.85.19.192/profound_bin/webProFound.exe). For all searches of mammalian sequences, a protein mass range between 5 and 150 kDa and peptide mass tolerance of 100 ppm were considered. Postsource decay (PSD) spectra were typically recorded in 7–12 segments, with each succeeding segment representing a 20% reduction in reflector voltage. About 50 shots were averaged per segment. Segments were pasted, calibrated, and smoothed under computer control by Bruker XMASS 5.0 software. The PSD spectra were interpreted manually.
µLC-Nano ESI Mass Spectrometry
The tryptic peptides were loaded onto a homemade capillary column (0.10 x 100 mm) packed with MAGIC C18 (5 µm, 200 Å) reversed phase resin (Michrom BioResources, Auburn, CA) and separated using a gradient from 5% MeCN/0.5% acetic acid to 40% MeCN/0.5% acetic acid for 50 min. The column was connected directly to an LCQDECA ion trap mass spectrometer (ThermoQuest, San Jose, CA) equipped with a nanoelectrospray ion source. The spray voltage was held at 1.2 kV and the tube lens potential was 10 V. The heated capillary was kept at 175°C with a voltage of 30 V. Full-scan spectra were recorded in positive mode over the mass range 350–2000 Da. Tandem mass spectrometry (MS/MS) data were automatically acquired on the two most intense precursor ions in each full-scan spectrum and searched against a self-built database containing mammalian proteins using Sequest software. The MS/MS spectra assignments were validated manually.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, we have analyzed pig oocyte protein patterns and their variations during in vitro maturation. In our culture system, porcine oocytes released from follicles at the GV stage initiated GV breakdown (GVBD) at 24 h, and by 28 h, most of the oocytes reached MI. Following this phase, the oocytes progressed through anaphase I/telophase I stage, and by 45 h, most reached the MII stage with the extruded typical first polar body (I PB) (Fig. 1). Based on morphological observations, nuclear maturation of oocytes under these culture conditions was synchronous and we were able to collect homogenous cell populations in different stages of in vitro maturation to prepare samples for 2-DE analyses reproducibly. The oocytes matured under these conditions had the capability of being penetrated at high rates (85% on average); however, the problem of polyspermy had an impact on the ultimate capability to reach blastocyst stage. The monospermic fertilization varied between 20% and 60% (average rate, 32%) and approximately 10%– 30% (15% on average) developed to the blastocyst stage when corrected by maturation rates (unpublished results).
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We applied a classical proteomics approach based on 2-DE protein separation coupled to protein identification by mass spectrometry on the samples of oocytes in different maturational stages. In the first phase of the study, many of the proteins of pig oocytes at the GV stage were identified and their position on 2-DE mapped. Due to the paucity of biological materials, such as oocytes, we preferred the miniformat (70 x 70 x 1 mm) of protein separation. Whole-cell extracts from 200 GV oocytes were separated on nonlinear pH 3–10 IPG strips and 12% SDS-PAGE was used in the second dimension, which resolved proteins over a mass range of 8–150 kDa. The PDQuest software complemented by visual inspection counted approximately 350 silver-stained protein spots. Selected spots (Fig. 2) were submitted to identification by mass spectrometry. The drawback of this core for proteomics 2-DE-based approach is that it requires relatively large amounts of sample even in the case of gels with a broad pH range. It is also difficult to obtain sufficient material to identify low-abundance proteins by mass spectrometry due to the relatively low sensitivity of total protein stain such as Coomassie blue. These problems can be partly overcome using recently developed, very sensitive fluorescence dyes. Furthermore, the staining intensity of these dyes has a broad dynamic range, with linear signal over four orders of magnitude, which improves protein quantification. Application of these technologies will be beneficial to the analyses of low-abundance cell populations, such as oocytes or stem cells, and the low abundance proteins present in such samples.
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The mass spectrometry-based protein identification approach, in general, uses two methods: peptide mass fingerprinting (PMF) and peptide sequencing. First, the protein is cleaved by a specific protease (usually trypsin) into peptides. For PMF, molecular masses of the peptides are typically determined by MALDI-MS. This information (peptide mass map) is used to search protein databases for protein identification. Protein identification by peptide sequencing is based on obtaining partial or complete sequence information by interpretation of peptide fragmentation spectra acquired by postsource decay (PSD) analysis on a MALDI-TOF mass spectrometer or by tandem mass spectrometry (MS/MS). Pig oocyte proteins were identified using the PMF approach. The peptide mass maps were measured by MALDI-MS following in-gel digestion of a protein with trypsin. Because the pig genome is far from complete, we were able to find only a few pig proteins. The majority of protein spots were identified from other mammalian species based on high sequence homology. To increase the probability of a successful protein hit and to confirm protein identity, we performed either a PSD or a MS/MS experiment on at least one peptide of each PMF-identified protein. Table 1 summarizes proteins that could be identified to date, measured position of protein spot on 2-DE in terms of molecular weight and isoelectric point, as well as PMF data. From the 35 protein spots listed in Table 1, only 18 protein spots represented individual proteins, and interestingly, there were 8 protein spots (SSP 0808, 1402, 1703, 1705, 2702, 2703, 2705, and 7702), each containing 2–3 different proteins, mostly due to the spreading of zona pellucida proteins. On the contrary, there were several proteins distributed in more than one spot (Table 1 and Fig. 2). While the protein spots SSP 5801 and 5804, identified as protein disulfide isomerase A3 as well as SSP 8401 and 8403, identified as glyceraldehyde 3-phosphate dehydrogenase, were each present in two closely located spots, suggesting the presence of isoforms or posttranslational modification, the distribution of the spots SSP 5806, 5702, and 6902 containing the major vault protein, or the spots 1802 and 1401 representing GRP94, indicated the possible cleavage of the protein with the presence of a lower molecular weight fragment.
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In addition, we sorted identified proteins according to their quantity to characterize which types of proteins belong to the most abundant in pig oocytes. However, for this purpose, we selected from all identified protein spots only those representing one protein. The results shown in Table 2 indicate clearly that the proteins such as peroxiredoxins, spermine synthase, and ubiquitin carboxyl-terminal hydrolase isozyme L1 belong to the extremely abundant oocyte proteins. Their levels are even higher or comparable with the level of beta-actin, usually the most abundant protein in somatic cells.
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Variation in Pig Oocyte Proteome During In Vitro Maturation
In the second phase of this study, we accomplished 2-DE analyses of porcine oocytes in MI and MII stages of in vitro maturation. The protein maps representing all stages of maturation, e.g., GV, MI, and MII, were computer evaluated and matched. The master gel of this matchset based on the sample of GV oocytes is shown in Figure 2. All previously identified protein spots have been present in all stages of oocyte in vitro maturation. The abundance of all but six (SSP 0808, 1703, 2702, 5804, 7701, and 1402) identified protein spots appeared to be stable in the course of maturation based on the calculation using a Student t-test implemented in PD Quest that does not reveal significant differences. From those six protein spots that underwent significant differential regulation in MI and MII compared with GV oocytes by a factor of at least two (Fig. 2, spots with SSP written in italics), five spots were the spots containing two or three proteins due to the presence of zona pellucida proteins; therefore, their quantitation was not adequate. Using PD Quest, we have selected other protein spots increased or decreased in MI and MII stages of in vitro maturation in comparison with GV oocytes. Identification of these proteins is currently under scrutiny in our laboratory.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Toward the Pig Oocyte Proteome
Current investigation of pig oocyte proteome and its changes during in vitro maturation has shed more light on the mechanisms of this process and opened the way to the molecular definition of pig oocyte maturation at the level of proteins. Nevertheless, it was evident that the presence of abundant proteins of zona pellucida on the surface of oocytes restricted quantitation of several protein spots on the mini gels. Zona pellucida sperm-binding proteins are sulfated and heavily O-glycosylated proteins, and this composition can explain their spreading through nearly the whole pI range and molecular sizes between approximately 50 and 70 kDa on 2-DE gels [19]. Furthermore, several proteins appeared to be fragmented. The reason for fragmentation of these proteins is not clear. In spite of this limitation of the 2-DE approach, this is the first pig oocyte proteome available so far that may be used as a reference map.
The proteins such as peroxiredoxins, which are involved in intracellular redox balance and protection against oxidative stress, belong to the highly abundant oocyte proteins. This finding indicates importance of antioxidant enzymes in porcine oocytes during the final stages of maturation. The expression of mRNA of Cu, Zn-superoxide dismutase, Mn-superoxide dismutase, and glutathione peroxidase in human and mouse oocytes was studied previously, and the results indicated the importance of the storage of these transcripts during oocyte maturation to allow successful embryo development [20]. We have demonstrated, for the first time, the maintenance of high levels of antioxidant enzymes, including several forms of peroxiredoxins and Mn-superoxide dismutase at the level of protein in porcine oocytes. Peroxiredoxins are thioredoxin-dependent peroxide reductases localized either in cytoplasm (PDX 1 and PDX 2) or mitochondria (PDX3). They represent new defense systems against reactive oxygen species, and their peroxidase activity relies on thioredoxin. In addition, peroxiredoxin enzymes might participate in the signaling cascades [21, 22]. Furthermore, we have found glutathione S-transferase Mu5 in relatively high levels in porcine oocytes. Glutathione S-transferases exist in multiple forms [23] and the Mu-class has been identified in mouse spermatogenic cells. They likely play a role in antioxidative protection [24].
Furthermore, two proteins identified as ubiquitin carboxyl-terminal hydrolase isozyme L1 (UBL1) and spermine synthase (SPSY) are present at extremely high levels in all stages of porcine oocyte in vitro maturation. It was shown previously that UBL1 is involved in toad oocyte maturation, possibly through an involvement in protein turnover and degradation [25]. In addition, it has been shown recently in somatic cells that regulation of ubiquitination has been associated with diverse proteasome-independent cellular functions [26]. Nevertheless, the high level of UBL1 that we have found in porcine oocytes might contribute to the precise timely regulated protein levels in transition from GV to MII stage of oocytes. Another highly expressed protein in porcine oocytes, spermine synthase, is involved in biosynthesis of spermine from spermidine. Using spermine synthase-deficient mice, it was shown that spermidine accumulation can explain increased resistance to oxidative stress, and this observation was the first indication that spermidine can serve as a free-radical scavenger [27]. Based on this observation, we suspect that high levels of spermine synthase in oocytes can be implicated in their susceptibility to oxidative damage. On the other hand, spermine, probably because of its high-affinity binding to DNA, is important for protection against chromatin damage [27].
Other groups of proteins identified in porcine oocytes can be classified according to their function as molecular chaperones, the proteins involved in energy metabolism, and members of the reductase/dehydrogenase family. We have identified mitochondrial HSP60 (SSP2803) and endoplasmic reticulum proteins calreticulin (SSP 0808), PDA3 (SSP 5801 and 5804), ER29 (SSP 7303), GRP78 (SSP 2804), and GRP94 (1802 and 1401) from the group of molecular chaperones. This family of proteins is implicated in correct folding of proteins and prevention of misfolding. While calreticulin, PDA3, GRP78, and GRP94 have been identified in oocytes of several species [28–30], the ER29 protein has not been found previously. More recently, Calvert et al. [30] reported identification of nine highly abundant molecular chaperones in the mouse egg proteome and, interestingly, their data suggested that these molecules localize to the oolema of the mature mouse egg. In addition to the chaperoning function, calreticulin is a major calcium storage protein in somatic cells, and its expression and regulation or localization in oocytes may play a crucial role in the tuning of calcium transience during oocyte maturation and fertilization [31].
We have also identified several proteins of energy metabolism. They include proteins of glycolytic pathway, alpha-enolase (SSP 7702), and triosephosphate isomerase (SSP 8201), mitochondrial ATP synthase beta chain (SSP 2702), dihydrolipoamide succinyltransferase component of 2-oxoglutarate dehydrogenase complex (SSP 6201), and galactokinase (SSP 5401). Altogether, the presence of these proteins might indicate that the oocytes are preparing for modification of energy metabolism following fertilization.
We have found three proteins belonging to the reductase/ dehydrogenase family. Among them, SSP number 7701, antiquitin (D7A1, SSP 7701) has been significantly increased in MI and MII stages compared with GV oocytes (Fig. 3). This protein is a member of the aldehyde dehydrogenase family, the enzymes catalyzing conversion of various aldehydes to the corresponding acids using the coenzymes NAD (+) or NADP (+). Antiquitin has been shown to be abundant in human ovary [32], and recent work of Rout and Armant [33] describing the expression of genes for alcohol and aldehyde dehydrogenases in mouse oocytes and preimplantation embryos has suggested the protective role of these enzymes against toxic effects of industrial pollutants as well as peroxidatic aldehydes generated during lipid peroxidation.
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Using PD Quest, we have selected several other proteins differentially regulated in MI and MII stages of in vitro maturation in comparison with GV oocytes. Identification of these proteins is currently under scrutiny in our laboratory, and these candidate proteins, together with the above-mentioned antiquitin, present potential markers of oocyte maturation and quality.
In conclusion, we have generated the proteome of porcine oocytes, including a list of proteins representing basic biochemical information about these germ cells. Some of the identified proteins were abundantly expressed during in vitro maturation of pig oocytes and may therefore play an important role in primary oocyte function—to undergo successful fertilization and to initiate zygotic development. Work is currently underway to characterize candidate proteins for differential regulation during in vitro maturation. We intend to perform similar proteome analysis of in vivo-matured oocytes and to compare selected candidate proteins during in vitro versus in vivo maturation. We expect that, for this purpose, the use of gel independent mass spectrometry analyses will be necessary in addition to gel-based technology. The major challenge for the future will be validation of the potential protein targets that will be selected from these studies and might be used as biomarkers of oocyte quality. This knowledge might be beneficial not only for basic science for improvement of oocyte culture conditions, which are still far from optimal, but also it may have implications for reproductive biotechnology. As a long-term goal, biomarkers identified in pig may be evaluated for other species, including humans.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Hana Kovarova, Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Rumburska str. 89, 277 21 Libechov, Czech Republic. FAX: 420 315 639 510; kovarova{at}iapg.cas.cz
Received: 29 March 2004.
First decision: 26 April 2004.
Accepted: 21 June 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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