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Unveiling the bovine embryo transcriptome during the maternal-to-embryonic transition

Department of Animal Sciences, Centre de Recherche en Biologie de la Reproduction, Pavillon Paul-Comtois, Laval University, Sainte-Foy, Quebec, Canada G1K 7P4¹ Laboratoire d'Organogenese Experimentale, LOEX, Hopital Saint-Sacrement, Sainte-Foy, Quebec, Canada G1S 4L8² Department of Human Genetics, Montreal Children's Hospital Research Institute, McGill University, Montreal, Quebec, Canada H3Z 2Z3

Correspondence should be addressed to M-A Sirard; Email: marc-andre.sirard{at}crbr.ulaval.ca

	Abstract

Top Abstract Introduction Results Discussion Materials and Methods Declaration of interest Acknowledgements References

 
Bovine early embryos are transcriptionally inactive and subsist through the initial developmental stages by the consumption of the maternal supplies provided by the oocyte until its own genome activation. In bovine, the activation of transcription occurs during the 8- to 16-cell stages and is associated with a phase called the maternal-to-embryonic transition (MET) where maternal mRNA are replaced by embryonic ones. Although the importance of the MET is well accepted, since its inhibition blocks embryonic development, very little is known about the transcripts expressed at this crucial step in embryogenesis. In this study, we generated and characterized a cDNA library enriched in embryonic transcripts expressed at the MET in bovine. Suppression subtractive hybridization followed by microarray hybridization was used to isolate more than 300 different transcripts overexpressed in untreated late eight-cell embryos compared with those treated with the transcriptional inhibitor, -amanitin. Validation by quantitative RT-PCR of 15 genes from this library revealed that they had remarkable consistency with the microarray data. The transcripts isolated in this cDNA library have an interesting composition in terms of molecular functions; the majority is involved in gene transcription, RNA processing, or protein biosynthesis, and some are potentially involved in the maintenance of pluripotency observed in embryos. This collection of genes associated with the MET is a novel and potent tool that will be helpful in the understanding of particular events such as the reprogramming of somatic cells by nuclear transfer or for the improvement of embryonic culture conditions.

	Introduction

Top Abstract Introduction Results Discussion Materials and Methods Declaration of interest Acknowledgements References

 
Early mammalian embryos exhibit very special features. They are produced by the fusion of two differentiated cells, the oocyte and the spermatozoa, to form a zygote, which then cleaves into an embryo composed of transcriptionally silent and undifferentiated cells. This transcriptionally quiescent embryo divides under maternal control until major genomic activation, a process that occurs at the 8- to 16-cell stage in bovine embryos (Camous et al. 1986, King et al. 1988, Frei et al. 1989, Kopecny et al. 1989, Telford et al. 1990). The substitution of maternally derived mRNA by the embryonic mRNA is called the maternal-to-embryonic transition (MET). This step in embryogenesis is crucial; inhibition of newly synthesized mRNA transcripts with -amanitin was shown to induce a developmental block in these embryos at the 16-cell stage (Plante et al. 1994, Liu & Foote 1997, Memili & First 1998).

Although the major transcriptional burst occurs in the 8- to 16-cell-stage embryos, minor transcriptional events have been reported at earlier stages (Barnes & First 1991, Plante et al. 1994, Hyttel et al. 1996, Viuff et al. 1996, Lavoir et al. 1997, Memili et al. 1998, Natale et al. 2000). Despite the fact that few transcripts are expressed at these initial embryonic stages, significant transcriptional activity and dramatic changes in the mRNA composition have been observed in six- to early eight-cell embryos (Natale et al. 2000). Furthermore, there is a striking distinction in the mRNA composition between pre- and post-MET embryos. Whereas the pre-MET embryos possess an mRNA population very similar to that of the oocyte, early eight-cell embryos already display an mRNA profile comparable with that found in the blastocyst (Natale et al. 2000). This suggests that as soon as the embryo reaches the MET, the embryonic program designed to bring it to the blastocyst stage is launched. This process includes an uncommon event called nuclear reprogramming, which is vital in establishing the fully pluripotent state found in embryos. The maintenance of pluripotency in early embryos is complex and not entirely understood yet. Some key genes, including POU class 5 homeobox 1 (POU5F1), Nanog homeobox (NANOG), signal transducer and activator of transcription 3 (acute-phase response factor) (STAT3), and sex determining region Y-box 2 (SOX2), are required to establish and maintain this event in embryos and embryonic stem cells, although the mechanisms involved in their activation and the identification of their downstream targets have not been fully elucidated (reviewed in Johnson et al. (2006)).

Several studies have described global mRNA expression patterns associated with the MET in mouse embryos (Sharov et al. 2003, Hamatani et al. 2004, Wang et al. 2004, Zeng et al. 2004, Li et al. 2006, Cui et al. 2007). It is obvious, however, that these patterns may differ from species to species. Recently, a global representation of the transcriptome found in bovine eight-cell embryos was obtained by probing microarray slides with amplified mRNA from the eight-cell-stage embryos treated or untreated with the transcriptional inhibitor -amanitin (Misirlioglu et al. 2006). Although this study contributed valuable information about transcription in bovine embryos at the MET, the commercial chip used was not enriched with embryonic transcripts, and therefore only provided a partial picture of genes expressed at that period. Previous studies using differential display RT-PCR (DDRT-PCR) also revealed the presence of transcripts differentially overexpressed in the eight-cell-stage bovine embryos compared with previous or subsequent stages of development (Natale et al. 2000, Ponsuksili et al. 2002, Tesfaye et al. 2003). Despite the success obtained with this PCR-based technique, the low sensitivity and poor efficiency in the recovery of the identified differentially expressed genes have limited this method's use.

In the present study, we used a suppression subtractive hybridization (SSH) technique to produce a library of newly transcribed genes in bovine eight-cell-stage embryos. This sensitive approach allowed us to identify more than 300 unique embryonic transcripts expressed at the MET. The high proportion of true-positive clones in our library was confirmed by quantitative RT-PCR. This library reveals that there is an unusually high representation of genes associated with transcription, RNA processing, and protein biosynthesis expressed at the bovine MET. The gene collection provided by this library offers a very useful tool for monitoring the onset of transcription in early embryos produced under different culture conditions or in cell reprogramming occurring in embryos produced by somatic cell nuclear transfer.

	Results

Top Abstract Introduction Results Discussion Materials and Methods Declaration of interest Acknowledgements References

 
Suppression subtractive hybridization and microarray hybridizations
Using SMART amplification and the SSH technology, we produced a library of more than 1000 clones overexpressed in control eight-cell embryos compared with -amanitin-treated eight-cell embryos. Spotting of the clones onto glass slides and hybridization of this microarray chip with probes produced from untreated and -amanitin-treated late eight-cell embryos revealed that 626 clones showed an expression increase that was twofold or greater in the untreated group.

Similar hybridizations were carried out with untreated and -amanitin-treated two- and four-cell embryos (data not shown). None of the 626 clones were demonstrated to be differentially expressed between the untreated and -amanitin-treated groups, signifying that there are no differentially expressed genes at either of these embryonic stages.

Sequence analysis of the hybridized clones corresponding to the embryonic expressed transcripts established that the 626 clones corresponded to 310 distinct transcripts. Closer examination of these 310 transcripts revealed the presence of 25 novel transcripts, 77 known but uncharacterized transcripts, and 208 known cDNAs with established function (Fig. 1A). The classification of the cDNAs with known function according to their primary molecular function following GO annotation produced an interesting blueprint of the early bovine embryo transcriptome at the MET (Fig. 1B). Indeed, 38% of the known cDNAs correspond to the genes involved in transcriptional regulation and RNA processing and an additional 18% correspond to the genes involved in protein biosynthesis. A selective list of these 310 transcripts, grouped according to their main molecular function following GO annotation and ranked by their overexpression, is displayed in Table 1. For a complete list of our 310 transcripts, see Supplementary Table 1, which can be viewed online at www.reproduction-online.org/supplemental.

View larger version (26K): [in this window] [in a new window]   Figure 1 Schematic representation of the 310 unique cDNA clones obtained following SSH and microarray hybridization. The absolute numbers of clones are indicated in parentheses. (A) The distribution of the cDNA clones according to the information available on their sequence. (B) The organization of transcripts with known function according to their primary molecular function following gene ontology annotation.

View larger version (42K): [in this window] [in a new window]   Figure 2 Quantification by real-time RT-PCR of the mRNA profile throughout bovine early development of 15 genes selected from our cDNA library. Stages are represented below histograms: GV, GV oocytes; 2c, 6-8c, and 8-12c represent embryos collected 32, 55, and 90 hpf respectively. Black histogram bars represent control embryos, while light grey histogram bars correspond to -amanitin-treated embryos. Each developmental stage was done in triplicate, and the amounts of mRNA shown represent the mean±S.E.M. of each transcript corrected with the GFP value obtained for each pool. *Significant difference (P<0.05). Only differences between -amanitin groups and their corresponding controls were displayed to lighten the figure.

To establish that our pools were not positively biased toward control embryos, the mRNA levels of two ubiquitous genes were measured (Fig. 3). Conserved helix–loop–helix ubiquitous kinase (CHUK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were identical in control and -amanitin groups confirming that the amount of maternal mRNA was unaffected by treatment with the inhibitor.

View larger version (16K): [in this window] [in a new window]   Figure 3 Quantification by real-time RT-PCR of housekeeping gene transcript levels throughout bovine early development. Stages are represented below histograms: GV, GV oocytes; 2c, 6-8c, and 8-12c represent embryos collected at 32, 55, and 90 hpf respectively. Black histogram bars represent control embryos, while light grey histogram bars correspond to -amanitin-treated embryos. Each developmental stage was done in triplicate, and the amounts of mRNA shown represent the mean±S.E.M. of each transcript corrected with the GFP value obtained for each pool. Different letters indicate a significant difference (P<0.05).

	Discussion

Top Abstract Introduction Results Discussion Materials and Methods Declaration of interest Acknowledgements References

 
In this study, we describe the construction of a subtracted library consisting of the bovine embryo transcriptome during the MET. SSH technology permitted the isolation of exclusively de novo-expressed genes in the late 8- to 12-cell embryos. Similar to the DDRT-PCR procedure, this technique detects differentially expressed genes between two groups, but with the advantages of significantly higher sensitivity and efficiency (Diatchenko et al. 1996). In our study, we identified 310 unique transcripts expressed de novo in the late eight-cell embryos. Our results also demonstrated the high specificity of our microarray studies, since the values obtained by quantitative RT-PCR for all the candidates screened were consistent with those generated through the microarray data analysis. Furthermore, these validated genes demonstrated a wide range of overexpression, from low to high differential expression, providing high confidence in the remaining transcripts present in the SSH. We feel certain that few false-positive clones will be discovered in the list generated by our microarray analysis. For the SSH validation, we used probes produced from a distinct pool of embryos and amplified by a different method (in vitro T7 transcription), and thus not the same material used in the construction of the library. Although this approach decreased the absolute discovery rate, it ensured low false-positive yields as it kept only the clones amplified differentially between the two treatments (-amanitin-treated versus untreated eight-cell embryos) by the two techniques (T7 and SMART PCR amplification), and thus reduced the bias caused by both methods.

The relevance of our library in terms of gene function was unexpected, as a very high proportion (56%) of our library was related to the genes involved in transcription, RNA processing, and protein biosynthesis. These findings support the prediction of elevated gene activity at the MET, and are consistent with a study in mouse, which reported a similar overrepresentation of transcripts coding for transcription factors and RNA processing factors among the transcripts expressed at the MET in the mouse two-cell-stage embryos (Zeng et al. 2004). Another recent study in mouse where a transcription factors array was hybridized with the mRNA extracted from mouse early embryos also showed a notable expression of transcripts coding for transcriptional regulators at the MET in mouse (Kageyama et al. 2006). One strength of SSH is the amplification of rare transcripts that often encode transcription factors and other regulatory proteins (Diatchenko et al. 1996). It is therefore not surprising that our procedure isolated a large number of transcriptional regulators compared with other techniques like DDRT-PCR that preferentially isolate highly expressed genes (Natale et al. 2000, Ponsuksili et al. 2002, Tesfaye et al. 2003).

Among the transcripts isolated in our library, only 6 genes out of 310 (nuclear transcription factor Y alpha (NFYA), H2A histone family, member Z (H2AFZ), acyl-CoA synthetase short-chain family member 1 (ACSS1, also known as ACAS2L), adenosylmethionine decarboxylase 1 (AMD1), ATP synthase, H+ transporting, mitochondrial F1 complex beta polypeptide (ATP5B), and nucleoside phosphorylase (NP)) were previously identified in a study comparing similar tissues through hybridization on commercial microarray chips (Affymetrix; Misirlioglu et al. 2006). This discrepancy may be attributed to the fact that many of our clones are not found in the Affymetrix GeneChip Bovine Genome Array gene annotation list or not yet assigned. Also, since only a fraction of the embryonic transcripts identified by Misirlioglu et al. (2006) was published, some of our clones may be present in the list of unpublished transcripts. Finally, differences between the embryo production systems and the precision in the developmental timing of the eight-cell embryos used may explain the variability between the two studies.

Interestingly, a significant proportion of the genes validated in the quantitative RT-PCR experiment (60%) was transcribed as early as 55 hpf, which corresponds to the eight- and very early eight-cell-stage embryos in our culture system. This early transcriptional activation is consistent with previous publications that have described the -amanitin-sensitive synthesis of proteins and the appearance of mRNA in this period of bovine embryogenesis (Barnes & First 1991, Natale et al. 2000). These findings of a significant proportion of transcripts expressed so early, before the documented MET, are also in agreement with the data published in mouse where many transcripts are expressed in the late one- and early two-cell-stage embryos, just before the major onset of transcription observed in the late two-cell-stage embryos (Hamatani et al. 2004). Several authors have speculated that a number of these early transcripts in bovine may play a key role in the activation of the major transcriptional burst detected at the 8- to 16-cell stage (Camous et al. 1986, King et al. 1988, Frei et al. 1989, Kopecny et al. 1989, Telford et al. 1990, Hyttel et al. 2000, Laurincik et al. 2003). This is likely to be the case for genes such as HNRNPA2B1, RBMX, KLF10, ZNF41, DDX5, and DDX39 which are related to transcription, either directly by binding DNA or indirectly through RNA processing (Franze et al. 1991, Venables et al. 2000, Carson et al. 2001, Rossow & Janknecht 2003, Kapadia et al. 2006, Subramaniam et al. 2007). To our knowledge, our study is the first to identify such a high proportion of transcripts expressed at the six- to early eight-cell embryos in bovine embryogenesis. If the results from these 15 profiles can be extrapolated to the remainder of the library, there may be many more genes expressed at the six-cell stage than previously imagined. This observation, combined with the presence of a relatively large number of transcriptional regulators and RNA processing factors in our library, suggests that many of these genes may play a role in the onset of the major transcriptional activation that occurs in the late eight-cell embryos.

Interestingly, our RT-PCR validation revealed a sudden and transitory expression at the six- or eight-cell stage for some genes (TP3, HNRNPA2B1, BECN1, and ZNF41). The transient expression of these genes suggests a punctual role for these factors in the early embryonic development during the MET. A transient expression at the MET was also observed for hundreds of transcripts in mouse two-cell-stage embryos suggesting a fundamental and specific role for these genes in this important transition from the maternal to the embryonic program experienced by early embryos at the MET (Hamatani et al. 2004, Zeng et al. 2004). To our knowledge, only one gene, EIF1, has been identified to have a similar transitory expression pattern in bovine embryos (De Sousa et al. 1998). EIF1 was absent from our library, although many eukaryotic translation initiation factors were detected, including EIF3A, EIF3H, EIF4G2, and EIF5. Its absence is most likely attributable to the fact that EIF1 is only present in bovine early eight-cell embryos (De Sousa et al. 1998) and late eight-cell embryos were used for the construction of our library. In the case of ZNF41, given its putative role in transcription (Franze et al. 1991), its transitory expression in the six- to eight-cell-stage embryos implies a role in gene transcription activation at the MET. Furthermore, its early transcription in the six-cell-stage embryo indicates that it could act as a precursor to the major burst of transcription detected in the late 8- and 16-cell-stage embryos. The sudden expression of the RNA-binding protein HNRNPA2B1, which is implicated in the trafficking and processing of mRNA (Landsberg et al. 2006), suggests that it may be involved in mRNA processing during embryonic genome activation.

Many unknown or poorly characterized genes in early embryogenesis were identified in our library. A large number of our clones demonstrated extremely high homology to these genes, such as the one coding for a putative spermatidal transition protein 3 (TP3) isolated from ram spermatids (Chevaillier et al. 1998). Transition nuclear proteins are known to replace somatic histones prior to their substitution by protamines during chromatin condensation in spermatogenesis (Meistrich et al. 2003). The presence of TP3 mRNA in oocytes, and its and transitory expression in the six-cell-stage bovine embryos is quite fascinating, but also puzzling. Because these proteins have not been associated with histone disruption during oocyte or embryo development, further studies are required to determine its function and to establish its involvement in events such as nuclear reprogramming in early embryos.

Unlike ZNF41 and HNRNPA2B1, the molecular function of BECN1, also known as ATG6, is not related to transcription. BECN1 is implicated in the autophagy process used by the cells to survive various stresses such as growth factor starvation or oxidative damage (Baehrecke 2005, Ferraro & Cecconi 2007). The autophagic process includes cytoplasmic degradation of cellular elements to eliminate affected constituents or to derive metabolites or energy from these degraded components. The short and sudden upsurge of BECN1 in the eight-cell embryos indicates that the culture medium may not have provided an optimal environment for embryonic growth. Some essential elements, such as growth factors, may have been excluded from the defined synthetic oviductal fluid (SOF) culture system (without serum) and caused the embryo to exploit its available resources to survive. However, these allegations are speculative and require further examination to be confirmed. Nevertheless, profiles for these genes illustrate the high potential of this library for the study and optimization of embryo culture systems, especially given the high sensitivity of embryos at the MET to suboptimal culture conditions (Camous et al. 1984). We believe that our spotted library of the MET transcriptome provides a novel and high-quality tool to monitor the health status of the early bovine embryo.

Several clones isolated from our library enhanced our understanding of the key regulators involved in the maintenance of pluripotency in the early embryos. In stem cells, the expression of ZFP42, also known as REX-1, is tightly regulated by NANOG, SOX2, and POU5F1, genes that are directly implicated in the maintenance of pluripotency in stem cells and embryos (Ben-Shushan et al. 1998, Du et al. 2001, Pelton et al. 2002, Hough et al. 2006, Shi et al. 2006, Masui et al. 2007). When ZFP42 activity is reduced in the mouse embryonic stem cells by RNAi knockdown, POU5F1 expression is perturbed and immediate differentiation of the cells is induced (kZhang et al. 2006). Our clone homologous to ZFP42 is completely absent from the pre-MET bovine embryos, while the post-MET embryos exhibited an increased expression. The simultaneous presence of ZFP42 and POU5F1 transcripts in bovine embryos (Kurosaka et al. 2004) suggests a role for ZFP42 in the conservation of pluripotency observed in bovine embryonic cells. Two other genes from our library have also been identified as regulators of POU5F1 and/or NANOG expression in embryonic stem cells: TPT1 and the GA-binding protein transcription factor alpha (GABPA; Kinoshita et al. 2007, Koziol et al. 2007). Similar to our observations for ZFP42, the mRNA expression profile in the bovine embryos of TPT1 is parallel to that of POU5F1 (Kurosaka et al. 2004). Finally, another transcript isolated by our SSH, the Kruppel-like factor 4 (KLF4), has been shown to act synergistically with POU5F1 in the transcription of genes in embryonic stem cells (Nakatake et al. 2006). KLF4 is also involved in stem cells renewal (Li et al. 2005b) and has been used with POU5F1 and other factors to reprogram somatic differentiated cells into pluripotent cells (Takahashi & Yamanaka 2006). KLF4 is expressed by the murine eight-cell embryos, as it was found in bovine in our library, despite the fact that murine embryos become transcriptionally active at the two-cell stage (Supplementary Table 1; Zeng et al. 2004). Therefore, instead of being expressed at the MET, KLF4 follows the expression pattern of crucial pluripotency genes: SOX2, POU5F1, and NANOG in mouse (Hamatani et al. 2004, Li et al. 2005a). A similar situation is observed with TPT1 that begins to be transcribed at moderate level at the four-cell stage in mouse and increases in subsequent stages, despite the fact that the embryonic genome is active at the two-cell stage (Supplementary Table 1; Hamatani et al. 2004). Therefore, KLF4 and TPT1 are expressed similarly in bovine and mouse in terms of embryonic stage with other genes involved in pluripotency regardless of the MET. The situation is different for GABPA (Kageyama et al. 2006) and ZFP42 (Supplementary Table 1; Hamatani et al. 2004) that are expressed at the MET in mouse, as in bovine, although the main factors involved in pluripotency are not yet expressed at this stage in mouse. This proposes a secondary role for these genes in embryogenesis, at least in mouse, than acting only in synergy with pluripotency factors like POU5F1 and NANOG.

Together, these results illustrate the depth of our library with respect to regulators involved in cell pluripotency and provide useful information for further studies of reprogramming in early embryos. Recent studies in mice have shown that aberrant gene expression is detectable as early as the two-cell stage in cloned embryos resulting from somatic cell nuclear transfer (SCNT), alluding to inadequate reprogramming of somatic cells during embryonic genome activation (Vassena et al. 2007). The bovine embryo represents a good model to study the consequences of SCNT because of its higher success rate compared with the mouse (Yang et al. 2007). Moreover, the longer time interval between nuclear transfer and genome activation permits a better cell reprogramming and a longer observation window. Therefore, the use of our library for microarray analysis represents a very powerful tool to study the SCNT and to monitor cellular reprogramming in cloned embryos compared with normally developing ones.

Our cDNA library provides an exceptional profile of the transcriptome at the MET in bovine embryos. More than 300 genes expressed in the late eight-cell-stage embryos cultured in vitro were isolated. A very high proportion of these genes have a demonstrated involvement in gene transcription or RNA processing. These findings are consistent with the high transcriptional activity presumed of the MET embryos. Since many of these transcripts were shown to be expressed in the six- and very early eight-cell-stage embryos, it is possible that some of these factors may act as precursors to the major transcriptional burst that occurs in the late 8- and 16-cell-stage embryos. In addition, since many regulators of cell pluripotency were also isolated in our library, this novel tool may be invaluable in the monitoring of nuclear reprogramming in somatic cell nuclear transfer experiments. Furthermore, the nature and composition of our library, which is spotted on cDNA microarray slides and available to researchers upon request, present a very powerful instrument for the study and development of new culture media for bovine embryos.

	Materials and Methods

Top Abstract Introduction Results Discussion Materials and Methods Declaration of interest Acknowledgements References

 
All chemicals were obtained from Sigma–Aldrich, unless otherwise stated.

Oocyte recovery and embryo production
Bovine ovaries were collected at a commercial slaughterhouse and transported in a 0.9% NaCl aqueous solution containing antimycotic agent. Cumulus–oocyte complexes (COCs) from 3 to 6 mm follicles were collected and only healthy COCs with at least five layers of cumulus cells were conserved for embryo production (Blondin & Sirard 1995).

COCs were put through in vitro maturation after three washes with HEPES-buffered tyrode lactate medium (TLH) supplemented with 0.3% fatty acid-free BSA (FAF-BSA; Lifeblood Medical, Adelphia, NJ, USA), 0.2 mM pyruvic acid, and 50 µg/ml gentamicin. Groups of ten COCs were placed in the droplets of media under mineral oil (M1180 from Sigma). Each droplet consisted of 50 µl maturation medium composed of modified (SOF; Holm et al. 1999) supplemented with 0.8% FAF-BSA, 1xMEM essential and nonessential amino acids (Gibco BRL), 1 mM glutamine, 50 µg/ml gentamicin, 0.1 µg/ml FSH, and 1 µg/ml 17β-estradiol. The droplets containing COCs were incubated in a humidified atmosphere for 24 h at 38.5 °C with 5% CO₂.

For IVF, five matured COCs were added to 48 µl droplets under mineral oil. These droplets were composed of modified TLH, supplemented with 0.6% FAF-BSA, 0.2 mM pyruvic acid, 10 µg/ml heparin, and 50 µg/ml gentamicin. Prior to transfer, the COCs were washed twice with TLH containing 0.3% FAF-BSA, 0.2 mM pyruvic acid, and 50 µg/ml gentamicin. Once transferred, 2 µl PHE (2 mM penicillamine, 1 mM hypotaurine, 250 mM epinephrine) were added to each droplet 10 min before the addition of semen. The semen consisted of a cryopreserved mixture of ejaculates from three bulls (Centre d'Insémination Artificielle du Québec; CIAQ, St-Hyacinthe, QC, Canada). The semen was thawed in 37 °C water for 1 min, put on a discontinuous Percoll gradient (2 ml of 45% Percoll over a 2 ml of 90% Percoll), and centrifuged at 700 g for 30 min at room temperature. The pellet was washed and centrifuged at 250 g for 5 min at room temperature. The supernatant was discarded and the spermatozoa were counted on a hemocytometer and resuspended in the IVF medium to obtain a concentration of 25x10⁶ cells/ml. Finally, 2 µl of the sperm suspension were added to each droplet and the incubation took place in a humidified atmosphere at 38.5 °C in 5% CO₂ for 18 h.

Following fertilization, presumed zygotes were mechanically denuded by repetitive pipetting, washed with PBS containing 0.3% FAF-BSA for complete removal of spermatozoa and cumulus cells, and then transferred to culture droplets (50 µl) in groups of 20–30 embryos. Embryo culture was performed in modified SOF1 medium (0.8% FAF-BSA, MEM nonessential amino acids, 1 mM glutamine, 1.5 mM glucose, 10 µM EDTA, and 50 µg/ml gentamicin) under mineral oil at 38.5 °C in 5% CO₂ in a reduced oxygen atmosphere (7%) with high humidity. In the -amanitin treatment groups, the embryos were cultured with 25 µg/ml -amanitin (A2263 from Sigma) and added to the culture medium from the zygote stage until embryo recovery. SOF1 medium was replaced after 72 h of culture with SOF2 medium (0.8% FAF-BSA, MEM essential and nonessential amino acids, 1 mM glutamine, 1.5 mM glucose, and 50 µg/ml gentamicin). The effectiveness of the defined SOF system for bovine in vitro embryos development has already been shown (Ali & Sirard 2002).

Oocyte and embryo collection
The 2-, 4-, 6- to 8- (early 8-cell), 8- to 12-cell (late 8-cell), and blastocyst-stage embryos were respectively collected 32, 44, 55, 90, and 8 days post-fertilization. For cDNA library construction, pools of 60 late eight-cell embryos cultured in the presence or absence of -amanitin were obtained. For microarray analysis, pools of ten embryos of 2-, 4-, and late 8-cell stages were gathered. Immature GV oocytes were collected for quantitative PCR analysis after selection and mechanical cumulus cell removal. Denuded oocytes were washed with RNAse-free PBS to ensure elimination of all cumulus cells and frozen in three pools of 20 oocytes at –80 °C until RNA extraction. Additionally, three pools of 20 embryos of two-, early eight-, late eight-, and blastocyst stages were collected for quantitative real-time RT-PCR experiments. All the embryos were washed three times with RNAse-free PBS, frozen, and stored at –80 °C until RNA extraction.

RNA extraction
RNA extraction was achieved using the PicoPure RNA isolation kit (Arcturus Molecular Devices, Sunnyvale, CA, USA), according to the manufacturer's instructions. RNA was eluted in 10 µl elution buffer and frozen at –80 °C until use. For real-time RT-PCR, 1 pg exogenous GFP poly(A) RNA was added to each sample at the beginning of the extraction as a technical exogenous control for RNA extraction and RT steps (Vigneault et al. 2004).

cDNA preparation and SSH
Total RNA from 60 -amanitin-treated and 60 untreated late eight-cell embryos was reverse transcribed and amplified using the Super SMART PCR cDNA synthesis kit (Clontech Laboratories), according to the manufacturer's instructions, for PCR-select cDNA subtraction applications. RNA integrity was monitored with the Bioanalyzer (Agilent Technologies, Mississauga, ON, Canada) before SMART amplification. Following the cDNA amplification optimization step, 21 PCR cycles for both samples provided satisfactory amplification yields without reaching a plateau. To avoid overcycling, both samples were removed after 19 cycles of amplification. Amplified double-stranded cDNA was then purified on a NucleoSpin Extract II column as recommended in the Super SMART instructions, and fractionated on a CHROMA SPIN-1000 column before being subjected to RsaI digestion. RsaI-digested cDNA was purified using the QIAquick PCR purification kit (Qiagen) and precipitated using sodium acetate as recommended. RsaI-digested cDNA was then dissolved in TBE buffer and the concentrations were determined using the NanoDrop ND-1000 spectrophotometer (NanoDrop technologies, Wilmington, DE, USA).

SSH was performed using the Clontech PCR-Select cDNA subtraction kit (Clontech), according to the manufacturer's recommendations. RsaI-digested cDNA from -amanitin-treated eight-cell embryos was the driver, and that of the untreated controls was the tester. Differentially expressed cDNA obtained from the SSH experiment was then cloned into the TOPO Cloning 5-min PCR cloning kit (Invitrogen) and transformed into One Shot MAX Efficiency DH5-T1 competent cells (Invitrogen).

Libraries production and cDNA slides preparation
Transformed bacteria were selected on LB/ampicillin/X-Gal agar plates. More than 1000 white colonies were picked and resuspended in 200 µl LB + ampicillin (50 µg/ml) and grown for 6 h at 37 °C with shaking. PCR amplifications were carried out from 2 µl bacterial suspension with the HotMaster Taq DNA polymerase (Eppendorf, Mississauga, ON, Canada) using the nested PCR Primer 1 (5'-TCGAGCGGCCGCCCGGGCAGGT-3') and 2R (5'-AGCGTGGTCGCGGCCGAGGT-3'; BD Biosciences, Mississauga, ON, Canada). The remaining bacterial suspension was kept in 20% glycerol at –80 °C. A 3 µl aliquot of each PCR was analyzed by electrophoresis on a 2% agarose gel to visualize single insert-containing clones.

The remaining PCR products were purified using Unifilter 384-well purification plates (Whatman, Toronto, ON, Canada). Aliquots from positive clones were retained for the sequencing reaction and clone identification. Purified PCR products were dried by SpeedVac and resuspended in H₂O/DMSO (1:1) before being spotted at three different locations on GAPS II glass slides (Corning, Lowell, MA, USA) using a VersArray ChipWriter Pro robot (Bio-Rad). The slides were then cross-linked with u.v. rays, according to the manufacturer's instructions.

mRNA amplification
Confirmation of the positive clones obtained from the SSH was performed by microarray hybridization of aRNA probes produced from new pools of ten late eight-cell-stage embryos treated and untreated with -amanitin. aRNA was produced using the RiboAmp HS RNA amplification kit (Arcturus Molecular Devices). This kit facilitates the production of 50 µg RNA from very small samples by two successive rounds of in vitro T7 RNA transcription. In the second round of transcription, regular UTPs were substituted with amino allyl UTPs (Arcturus Molecular Devices) to allow subsequent labeling of the aRNA.

Labeling of aRNA
From each sample (-amanitin-treated and untreated), 2.5 µg aRNA were used for labeling. Probes were labeled with Alexa Fluor 555 and 647 reactive dye packs (Molecular Probes, subdivision of Invitrogen), according to the manufacturer's protocol. Alexa-coupled aRNA was purified with PicoPure RNA isolation kit (Arcturus) to remove uncoupled dyes.

Microarray hybridization
Hybridization was performed at 50 °C for 18 h in SlideHyb Glass Array Hybridization Buffer #1 (Ambion, Austin, TX, USA) using a Slide Booster apparatus (Advalytix, Concord, MA, USA). To remove unhybridized probes, the slides were washed twice with 1xSSC/0.2% SDS for 15 min at 50 °C and twice with 0.1x SSC/0.2% SDS for 15 min at 50 °C. Then the slides were scanned using the VersArray ChipReader System (Bio-Rad Laboratories) and analyzed with the Array-Pro Analyzer software (Media Cybernetics, San Diego, CA, USA). A dye-swap experiment was conducted to reduce the negative effects resulting from the differences in dye incorporation and slide reading.

Microarray analysis
Microarray data analysis was performed using the NIA Array Analysis software, a web-based tool accessible at the following address: http://lgsun.grc.nia.nih.gov/ANOVA/ (Sharov et al. 2005). Only clones with a significant signal and with at least a twofold change (false discovery rate (FDR)<0.05) in the untreated versus -amanitin-treated sample were considered to be positive and subsequently sequenced.

Additional experiments were carried out with untreated and -amanitin-treated two- and four-cell-stage embryos. All procedures, from RNA extraction to microarray hybridization and data analysis, were identical to those described for the eight-cell embryos.

Sequencing and clone identification
DNA sequencing was performed using an automated ABI 3730 DNA sequencer (PE Applied BioSystems, Streetsville, ON, Canada). Sequencing reactions were carried out with the ABI Prism BigDye Terminator Cycle Sequencing Ready Reactions kit (PE Applied BioSystems) and nested PCR primer 1 (5'-TCGAGCGGCCGCCCGGGCAGGT-3'; BD Biosciences). Sequence traces were visualized with the freeware Chromas 1.45 (Technelysium Pty Ltd, Tewantin, Queensland, Australia) and uploaded into a cDNA Library Manager (program written by Genome Canada Bioinformatics Help Desk according to our needs; Vallee et al. 2005). The cDNA Library Manager automates and facilitates the sequence trace analysis and clone identification. Briefly, the sequence traces were uploaded into the cDNA Library Manager under a particular library name and clone number. The sequences were then trimmed (Phred software) and compared against a locally installed GenBank database (http://www.ncbi.nlm.gov/BLAST/). BLAST results, including GenBank accession number, UniGene number, score, e-value, % homology, number of nucleotides aligned, position of alignment, etc. were compiled into a report.

Gene ontology annotation
Gene ontology (GO) annotation of the genes transcribed by the eight-cell-stage embryos was performed using Onto-Miner from Onto-tools available online at http://vortex.cs.wayne.edu/projects.html (Khatri et al. 2004). The transcripts that did not generate annotation results were annotated individually using the AmiGO tool provided by the GO Consortium, available online at http://www.geneontology.org (Harris et al. 2004). The transcripts were regrouped according to their molecular functions using broad functional terms to limit the number of categories.

cDNA preparation and quantitative RT-PCR
mRNA from pools of 20 oocytes and embryos of 2-cell, 6- to 8-cell, 8- to 12-cell, and blastocyst stages was reverse transcribed using oligo(dT)₁₈ primers (1 µM; or random nanomers (10 µM) in a confirmation experiment) and Sensiscript RT kit (Qiagen), following the manufacturer's recommendations, with the addition of 10 units of recombinant RNasin (Promega). The primers used for real-time RT-PCR are listed in Table 2, and were designed using the Primer3 web interface (Rozen & Skaletsky 2000; available at http://frodo.wi.mit.edu/) from the sequences obtained from the sequencing of the positive clones obtained by microarray analysis. For each gene examined, a standard curve consisting of PCR products purified with the QIAquick PCR purification kit (Qiagen) and quantified by NanoDrop was included. Real-time PCR was performed on a LightCycler apparatus (Roche Diagnostics, Laval, QC, Canada) using SYBR green incorporation. Each reaction contained the cDNA corresponding to half of an oocyte or embryo and a mixture containing 0.5 µl of each primer (10 µM), 1.6 µl of 25 mM MgCl₂ (final concentration of 3 mM), 2 µl of the SYBR green mix containing dNTPs, FastStart DNA polymerase enzyme, and buffer (Roche). The PCR conditions used for all genes were as follows: denaturing cycle of 10 min at 95 °C; 45 PCR cycles (denaturing, 95 °C for 1 s; annealing, 58 °C for 5 s; extension, 72 °C for 10 s); a melting cycle consisting of 95 °C for 1 s, 70 °C for 30 s, and of a step cycle starting at 70 °C up to 95 °C with a 0.2 °C/s transition rate; and a final cooling cycle of 40 °C for 30 s. The cDNA quantification was performed using LightCycler Software Version 3.5 (Roche) by comparison with the standard curve. PCR specificity was confirmed by melting curve analysis and sequencing of the PCR product.

	Declaration of interest

Top Abstract Introduction Results Discussion Materials and Methods Declaration of interest Acknowledgements References

 
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

	Funding

 
The authors would like to thank Natural Sciences and Engineering Research Council of Canada and Fonds québécois de la recherche sur la nature et les technologies for funding this study.

	Acknowledgements

Top Abstract Introduction Results Discussion Materials and Methods Declaration of interest Acknowledgements References

 
We thank Isabelle Laflamme for her technical assistance in embryo production and Dr Isabelle Dufort for her assistance with microarray hybridizations. We also thank Dr Serge McGraw and Dr Andrea K. Lawrance for manuscript revision and correction.

Received February 20, 2008
First decision March 19, 2008
Revised manuscript received September 16, 2008
Accepted November 5, 2008

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