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Gamete Biology |
a Physiologie de la Reproduction et des Comportements, UMR 6073 Institut National de la Recherche Agronomique/Centre National de la Recherche Scientifique/Université François Rabelais de Tours, F-37380 Nouzilly, France
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gamete biology, gene regulation, meiosis, oocyte development
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Detailed morphological descriptions have been essential in delineating successive stages of transcriptional activity during bovine oogenesis. Ultrastructural observation of the nucleoli, with subsequent corroboration by immunocytochemical localization of RNA polymerase I and nucleoli labeling following incorporation of radioactive uridine, have provided a precise chronology of transcription of ribosomal genes (reviewed in [2]). Such detailed analysis is not available for transcription of heteronuclear RNA, the precursor for small nuclear and messenger RNA. However, radioactive labeling of the nucleoplasm has provided useful information. In the primordial follicle, the oocyte appears transcriptionally quiescent. The synthesis of both ribosomal and heteronuclear RNA is first detected in the secondary follicle. Transcription becomes extremely intense within the tertiary follicle up until an oocyte diameter of 100 µm. It decreases thereafter and eventually returns to an undetectable level. GVBD and meiotic resumption are preceded by a short burst of transcriptional activity, as evidenced by radioactive uridine incorporation into RNA [3–5]. This transcription seems functionally important because its inhibition by 
-amanitin (an RNA polymerase II inhibitor) impairs the maturation process. Although informative, these traditional approaches for the study of global transcriptional activity are of low sensitivity and shed little light on the nature of the expressed genes.
Reverse transcription coupled with polymerase chain reaction (PCR) allows detection of rare messengers in small biological samples and has therefore been the method of choice for studying individual genes in oocytes. Altogether, the expression of 100–150 genes has been demonstrated in bovine oocytes. This technique has been applied to evaluate the expression of genes involved in biological functions such as cell communication [6], metabolism [7–9], stress response [10], or apoptosis [11]. However, using such an approach for even relative quantification requires a tedious preliminary experimental design for each gene.
Over the past decade, in parallel with progress in genome sequencing of several model species, methods such as hybridization of cDNA populations onto arrayed libraries of preidentified expressed sequence tags (EST) have been developed to characterize broad-scale transcription patterns. Although an obvious limitation of this approach is that no novel gene can be isolated, it represents a time- and cost-effective method of screening the expression of hundreds to thousands of known genes in a single experiment. Until recently, due in part to the requirement for microgram quantities of RNA, this technology did not appear appropriate for the study of mammalian oocytes. Only the most recent technological advances, i.e., linear amplification of the cDNA population, have made it possible to consider expression profiling in this biological model of extremely limited availability.
In this study, we have compared gene expression patterns in bovine oocytes before and after in vitro maturation using heterologous cDNA array hybridization: bovine cDNA probes were hybridized onto arrays of human EST. The three main objectives of our experiment were to validate this technique applied to oocytes, to characterize a transcription pattern in this model, and to identify genes whose transcripts are differentially regulated during maturation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Oocyte-cumulus complexes (OCC) were collected by aspiration from follicles 3–8 mm in diameter on bovine ovaries obtained from a local slaughterhouse, as previously described. OCC presenting a dense, compact cumulus and a homogeneous oocyte cytoplasm were selected and washed several times in modified phosphate-buffered saline solution (for details see [12]). Approximately two thirds of the OCC were submitted to mechanical treatment until separation of oocytes from surrounding somatic cells. After several washes, absence of cumulus cell was checked visually under a stereomicroscope and later confirmed by Hoechst 33258 chromatin staining of 10 randomly selected oocytes. Denuded oocytes were separated into two groups (groups 1 and 2) and frozen at -80°C until RNA isolation. The remaining third of the OCC were allowed to undergo in vitro maturation for 24 h at 39°C in water-saturated air with 5% carbon dioxide; groups of 50 OCC were incubated in four-well plates (Nunc, Roskilde, Denmark) in 500 µl Tissue Culture Media 199 (Sigma, St. Louis, MO) supplemented with 10 ng/ml epidermal growth factor (EGF) (Sigma). These conditions routinely result in over 90% nuclear maturation rate and good embryonic development potential [13]. OCC with an expanded cumulus were selected, and oocytes were denuded as described and frozen (group 3).
Isolation of RNA
Total RNA from the three oocyte groups was isolated in parallel using the Tripure Isolation Reagent (Roche Diagnostics, Mannheim, Germany) following the manufacturer's instructions. As a control for RNA extraction and absence of contaminating genomic DNA, an aliquot was reverse transcribed (RT) and submitted to PCR. Specific primers for the cell-cycle control genes cyclin B1 (CCNB1) and cell division cycle 2 (CDC2), designed to hybridize within two exons separated by a short intron, were used. Only short fragments, amplified from cDNA template, could be detected.
The whole process (oocyte collection, in vitro maturation, RNA isolation, and control RT-PCR reaction) was repeated with four different batches of ovaries. Only at the end was RNA from each collection pooled, leading to total RNA amounts equivalent to populations of 220 oocytes per group.
Probe Generation
The 32P-labeled probes were generated from this RNA using the Atlas-Smart system (Clontech Laboratories, Palo Alto, CA) following the manufacturer's protocol with minor modifications. Briefly, an RNA amount equivalent to 220 oocytes from each group was reverse transcribed using the SmartII and CDS primers (Smart PCR cDNA synthesis kit; Clontech Laboratories). Actual cDNA synthesis was confirmed by subjecting aliquots of each reaction mix to amplification by PCR with specific primers for the CCNB1, CDC2, and ß-actin genes. Each cDNA population was then amplified using the Atlas Smart probe amplification kit (Clontech Laboratories). A sample was subjected to PCR amplification and aliquots were analyzed on an agarose gel after 15, 18, 21, 24, and 27 cycles. Saturation was reached after 18 cycles; the optimal number of PCR cycles to be used for subsequent probe preparation was deduced to be 17. Amplified cDNA was purified and the concentration estimated based on optical density. Five hundred nanograms from each cDNA mix was used as a substrate for random prime labeling (Atlas Smart probe amplification kit) in the presence of [-32P]dATP (Perkin Elmer Life Sciences, Boston, MA). Finally, radiolabeled probes were purified as recommended. Radioactivity was estimated by scintillation counting.
Array Hybridization
Broad-scale expression profiling was performed using the Atlas human 1.2 cDNA expression array (Clontech Laboratories). These nylon membranes are spotted with 1176 human cDNA fragments from known genes between 200 and 600 base pairs in length. Spots are arrayed onto six regions according to gene function: A) oncogenes, tumor suppressors, cell cycle regulators; B) transporters, signal transduction; C) GDP/GTP exchangers, GTPase stimulators and inhibitors, apoptosis; D) transcription factors, cell signaling, and extracellular communication; E) cell surface antigens, cell adhesion, receptors; F) stress response, cell-cell communication. In addition, cDNA fragments from selected housekeeping genes, human genomic DNA, phage and plasmidic DNA are spotted as positive and negative controls. The manufacturer's instructions were followed with minor modifications. After heat denaturation, probes generated from oocyte groups 1–3 were hybridized at 68°C overnight to one of three identical membranes (accordingly numbered 1–3). After several washes at 65°C in 2x saline-sodium citrate (SSC), the membranes were exposed to a phosphor storage screen (Amersham Biosciences, Orsay, France) for 3 days, then 14 days before image acquisition by a Storm 840 (Amersham Biosciences).
Gene Expression Analysis
Relative radioactive intensity was measured using the ImageQuant software (Amersham Biosciences). Intensity was calculated in areas surrounding spots visible on any of the three membrane images, corrected for the local average background. Most visible spots were in the linear range after a 14-day exposure, with the exception of spots at positions Ah2, Aj13, Aj14, which could be quantified after the shorter exposure, and Fj13 and G11, which had already reached saturation at that point. Differences between the membranes appeared in the global hybridization intensity as well as an irregular background. To correct for these variations, the sum of the intensities of the spots within the considered region (cDNAs are arranged in the array in six regions labeled A–F) was normalized between the membranes. Normalized values were then compared between the membranes. Comparison of membranes 1 and 2, hybridized with probes generated from duplicate immature oocyte groups 1 and 2, allowed for control of experimental reproducibility. Comparison of membranes 1 and 2 with membrane 3 allowed for identification of differentially expressed genes following in vitro maturation.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We then proceeded with quantitative analysis. We considered the abundance of each transcript, i.e., its proportion within the entire population. The variation factor, defined as the ratio of intensities before IVM (membrane 1 or 2) and after IVM (membrane 3) corrected for normalization between the membranes (see Materials and Methods), was calculated for each hybridization signal. The signal at position Fj13 was out of linear response range, so its variation factor could not be estimated. A graphical representation of variation factors calculated based on membranes 1 and 2 is presented in Figure 1. The global correlation coefficient was 0.993. The threshold for significant variation was conservatively set at twofold. Using this criterion, genes were sorted into three categories based on their variation factor: those for which the relative transcript level decreased, increased, or was not significantly affected. Over 80% of the genes (244 out of 300) exhibited a similar variation tendency (decrease, increase, or stability) in our duplicate experiment. Divergences between the data usually corresponded to "frontier" values on one of the membranes with regard to our twofold threshold for variation and/or a very weak signal or a high local background that hindered quantification. Among these, only two genes (out of 300, i.e., 0.7%) were found to display opposite variations depending on the membrane considered. Thirty-seven and 33 genes (13% and 11% of those detected) consistently displayed a lower and higher relative transcript level respectively after IVM.
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Over 300 signals could be detected. Based on the tentative functional classification proposed by the membrane's manufacturer (see Materials and Methods), the distribution of the corresponding transcripts is represented in Figure 2. A roughly similar number of genes in each category (approximately 50) could be detected, with the relative expression level of most of them (between 28 and 42) appearing stable. The corresponding genes are listed in Tables 1–6 . In addition, Table 7 corresponds to control housekeeping genes. Finally, genes whose transcript displayed a reproducible twofold decrease or increase of their relative abundance are listed in Tables 8 and 9, respectively. For each gene, the Homo sapiens official or interim (with an asterisk) gene symbol and name (HUGO Gene Nomenclature Committee [HGNC]) is indicated as well as more common alternate symbols and names in italic letters.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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At the outset, it was necessary to determine whether cDNA array hybridization is an appropriate technique to analyze gene expression and its regulation in bovine oocytes. Because no membranes spotted with cDNA fragments derived from bovine genes are commercially available, we undertook a heterologous screening using nylon membranes spotted with human cDNA fragments. Our differential model was the bovine oocyte before and after in vitro maturation. To our knowledge, cDNA macroarrays have not been used with probes generated from oocytes, and we had to design a protocol for this cellular model. A point of particular importance was the number of oocytes required for the experiment. Bovine oocyte total RNA content has been estimated between 2 and 3 ng [14]. According to the manufacturer's recommendation, the RNA requirement to synthesize a probe with the conventional protocol is 2–5 µg, which would represent as many as 2500 oocytes. Alternatively, 50 ng total RNA (corresponding to 25 oocytes) would be sufficient if the probe was generated by linear PCR amplification. On the other hand, with a lower number of oocytes, stronger influence of individual variations in gene expression might be expected. We balanced the practical and statistical criteria and generated each probe from a total of 220 oocytes.
Compared with usual DNA array experiments, heterologous screening presents additional limitations. First, random primers were chosen to generate the probes rather than CDS primers, targeted to human cDNA fragments spotted onto the membrane, but whose complementary sequence might not have been conserved in the bovine. Such highly complex probes are expected to result in decreased sensitivity and higher background. Second, to allow for probe hybridization despite imperfect sequence conservation between the two species, arrangements of cDNA fragments several hundred base pairs long were preferred over oligonucleotide arrays, at the cost of decreased specificity. However, gene misidentification resulting from potential hybridization of a bovine cDNA fragment from a different gene was expected to remain a rare event. Reciprocally, it is more likely that, because of local sequence divergence, some genes actually expressed in bovine oocytes do not result in a positive signal.
Overall, 300 spots (26%) could be detected over the background out of 1176 present on the array (not considering housekeeping genes). This proportion falls into the 20%–70% range expected for Atlas arrays, showing that the level of sequence homology between human and bovine genes is compatible with heterologous cDNA array screening. In addition, our relatively low percentage supports the specificity of hybridization.
After quantitative analysis, using a twofold ratio as the threshold for considering variation during IVM, over 80% of the genes exhibited a similar variation tendency (decrease, increase, or stability) in our duplicate experiment. Correlation between variation factors calculated based on membranes 1 and 2 was found to be 0.993. This value reflects good reproducibility of probe generation and hybridization and guarantees that occurrence of artifact signals remains limited.
Gene Expression Profile
Over 300 spots could be detected, leading to the largest transcription profile available so far in the bovine oocyte. The corresponding genes are known to be involved in a variety of biological functions. Overall, this broad range expression pattern indicates that the oocyte possesses the potential to accomplish most cellular processes, and this potential is later transmitted to the embryo after fertilization.
Expression at the messenger RNA or protein level of only a minority of these genes had previously been analyzed in cattle oocytes or zygotes. We confirmed the presence of transcripts for TGFB2 [15], ERBB3 [16], HSP70.1 [17, 18], GPI [7], Cu/Zn SOD [19, 20], and HDAC1 [21]. In agreement with published data, EGF did not appear to be expressed [15]. In addition, we were able to detect transcripts for the IGFBP1, TOP1, and GSTA2 genes, whereas the respective corresponding transcript (in one-cell zygotes) and proteins had failed to be detected in previous studies [22–24]. On the other hand, a gene known to be expressed in bovine oocytes may not be detected. For example, messengers for IGFBP2, IGFBP3 [22], IGF1R, IGF2R [15], RARB [25], p53 [11], or the Na/K ATPase 1 chain [26] were previously shown by RT-PCR to be present in bovine oocytes or zygotes, yet they could not be evidenced in our screening. Two hypotheses can account for these discrepancies: either a very low copy number or a sequence divergence between the bovine gene and human EST spotted onto the membrane. Nonetheless, these examples underline that absence of a signal on all three membranes should not be interpreted as total repression of the corresponding gene. The profile of 300 genes presented here should therefore be taken as a minimal one.
The expression of most genes identified in the present study has not previously been investigated in bovine oocytes. Comparison with an expression profile in mouse oocytes [27] reveals 53 common transcripts. A similar comparison with the serial analysis of gene expression tags catalog available for human oocytes [28] proved difficult because the authors did not employ the HGNC nomenclature; as a result, only 15 common transcripts could be positively identified. However, when a bovine oocyte messenger RNA was not found to be conserved in mouse or human, in most instances related transcripts could be identified, such as different members within a given family or different subunits of a given complex. This establishes a correlation between oocyte transcriptomes of various mammalian species. It also indicates that the various transcriptome analysis methods are complementary rather than redundant.
Variation During Maturation
A large proportion of the transcripts stored within the oocyte cytoplasm are believed to disappear during maturation, as observed in Xenopus and mouse [29, 30]. Therefore, housekeeping genes, which are often used for normalization between samples, might not be an appropriate reference for maturing oocytes. In this particular model, hybridization onto cDNA array can only measure the variation of relative abundance of individual transcripts. This has to be kept in mind when analyzing our differential expression screening. This majority of transcripts that do not appear affected actually must have followed the global stream of destabilization, while genes that exhibit a lower relative transcript level after IVM underwent an even more drastic destabilization. Degradation may be coupled with active translation that occurs during oocyte maturation and results in changes in protein synthesis pattern [31]. Finally, the genes that display a higher relative transcript level after IVM may either only resist destabilization better or be actually transcribed during early maturation. Only an analysis of individual genes by a quantitative or semiquantitative method will provide a definitive answer. Despite this limitation, DNA array screening remains a powerful tool for analysis of gene expression. In a single experiment, we generated a list of 70 transcripts as candidates for differential regulation during oocyte maturation. Respectively, 37 and 33 transcripts (listed in Tables 8 and 9) displayed a reproducible twofold decrease and increase of their relative abundance. Together they form a molecular signature of oocyte cytoplasmic maturation in vitro. We are aware that, despite good experimental care, some differences in hybridization signals may be attributable to technical problems such as local poor hybridization or washing. Complementary expression analysis of several among these candidates by alternative techniques (reverse-transcription coupled with semiquantitative or real-time PCR) will be necessary to confirm them as markers of oocyte maturation.
A central feature of oocyte maturation is the transient reactivation of the cell cycle that had remained arrested during the entire course of follicular growth. During maturation, the oocyte also prepares for a dramatic modification of its environment and energy metabolism following ovulation and fertilization. Embryonic developmental potential will also depend on the ability to sustain DNA replication and repair and to ensure apoptosis in cells bearing chromosomal abnormalities [32]. Several transcripts identified in our differential screening are involved in these processes.
Several cell cycle-related genes appear among our candidates. CCNB1 and CDC2 encode subunits of the M-phase promoting factor, a critical component of meiotic resumption. CDC2 and CCNB1 relative abundances decreased and increased, respectively. CCNB1 is targeted for degradation by the proteasome by the ubiquitin conjugating enzyme UBE2C, whose transcript relative level also increased during maturation. The observed lower relative abundance of two peroxiredoxin isoforms (PRDX1 and 2) and a modified pattern of glutathione S-tranferase isoenzymes representation (decrease of GSTT1 and GSTM1 and increase of GSTA2 mRNA relative levels) may be related to regulation of oxidative stress. DNA polymerase beta (POLB) was identified among several transcripts displaying increased relative abundance after maturation and involved in DNA replication or repair. In agreement with this observation, an estrogenic upregulation of POLB protein following LH surge in oocytes of preovulatory ovine follicles has been demonstrated in parallel with increased developmental competence of the oocytes [33]. Finally, several transcripts of genes known to trigger or prevent apoptosis were found to be differentially regulated in these experiments (DAD1, CASP4, FASTK, BCL2L1).
In conclusion, thanks to PCR linear amplification of the entire cDNA population, we were able to generate a transcription profile of bovine oocytes and a list of candidate genes for differential regulation during maturation, starting with very small amounts of biological material. In these initial experiments, we conservatively used 200 oocytes to generate each probe. In fact, fewer oocytes should be sufficient and will be used in future experiments. We now plan to compare transcription profiles in oocytes at various stages during folliculogenesis as well as expression of selected genes during in vivo vs. in vitro maturation. These future experiments will provide important data regarding oocyte differentiation and gradual acquisition of the ability to resume meiosis, reach the metaphase II stage, be fertilized, and sustain early embryo development at least until the embryonic genome takes over. Some of these transcripts may be used as markers of oocyte quality for the improvement of oocyte culture conditions by addition of supplements of biological or synthetic origin. Similar transcript levels in in vitro- and in vivo-matured oocytes should indicate adequate culture conditions. These observation will then be available for transposition to other mammalian species, including human.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 33 247 42 77 43; dalbies{at}tours.inra.fr
Received: 29 May 2002.
First decision: 21 June 2002.
Accepted: 7 August 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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