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Selective degradation of maternal and embryonic transcripts in in vitro produced bovine oocytes and embryos using sequence specific double-stranded RNA

Institute of Animal Science, Animal Breeding and Husbandry Group, University of Bonn, Endenicher Allee 15, 53115, Bonn, Germany and ¹ University of Veterinary Medicine Vienna, Veterinär platz 1, A-1210, Vienna, Austria

Correspondence should be addressed to D Tesfaye; Email: tesfaye{at}itz.uni-bonn.de

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
RNA interference (RNAi) has been used for selective degradation of an mRNA transcript or inhibiting its translation to a functional protein in various species. Here, we applied the RNAi approach to suppress the expression of the maternal transcript C-mos and embryonic transcripts Oct-4 in bovine oocytes and embryos respectively, using microinjection of sequence-specific double-stranded RNA (dsRNA). For this, 435 bp C-mos and 341 bp Oct-4 dsRNA were synthesized and microinjected into the cytoplasm of immature oocytes and zygotes respectively. In experiment 1, immature oocytes were categorized into three groups: those injected with C-mos dsRNA, RNase-free water and uninjected controls. In experiment 2, in vitro produced zygotes were categorized into three groups: those injected with Oct-4 dsRNA, RNase-free water and uninjected controls. The developmental phenotypes, the level of mRNA and protein expression were investigated after treatment in both experiments. Microinjection of C-mos dsRNA has resulted in 70% reduction of C-mos transcript after maturation compared to the water-injected and uninjected controls (P<0.01). Microinjection of zygotes with Oct-4 dsRNA has resulted in 72% reduction in transcript abundance at the blastocyst stage compared to the uninjected control zygotes (P<0.01). Moreover, a significant reduction in the number of inner cell mass (ICM) cells was observed in Oct-4 dsRNA-injected embryos compared to the other groups. From oocytes injected with C-mos dsRNA, 60% showed the extrusion of the first polar body compared to 50% in water-injected and 44% in uninjected controls. Moreover, only oocytes injected with C-mos dsRNA showed spontaneous activation. In conclusion, our results demonstrated that sequence-specific dsRNA can be used to knockdown maternal or embryonic transcripts in bovine embryogenesis.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Presently, the genomes of various species including the bovine are largely sequenced. Moreover, several studies have been carried out during the last decade to investigate the expression patterns of genes in bovine embryogenesis in response to the various culture and treatment conditions (Rizos et al. 2002, 2003, Lonergan et al. 2003a, 2003b, Tesfaye et al. 2004, El-Halawany et al. 2005). Despite this fact, the function of a large number of genes in mammalian embryogenesis is not yet investigated or not known. Transgenic and gene-targeted mouse lines have been used extensively to study the function of various genes in mammalian embryogenesis. These mouse knockout studies are relatively slow and this tool cannot keep pace with the rapid accumulation of new sequence information produced by the various genome projects. Therefore, what is needed is a technique that can be used to jump directly from sequence to function in a whole animal. For this, the post-transcriptional gene silencing (PTGS) by double-stranded RNA (dsRNA) or RNA interference (RNAi) has emerged as a new tool for studying gene function in an increasing number of organisms (Fire 1999). Following its application in Caenorhabditis elegance (Fire et al. 1998), the RNAi approach by introduction of dsRNA has been used in various mammalian species such as human (Brusselmans et al. 2005, Cheng et al. 2005, Hyslop et al. 2005), mouse (Svoboda et al. 2000, Wianny & Zernicka-Goetz 2000, Grabarek et al. 2002, Ma et al. 2002, Stein et al. 2003, Alizadeh et al. 2005, Gui & Joyce 2005, Plusa et al. 2005) and porcine (Cabot & Prather 2003). Most recently, the work by our group (Nganvongpanit et al. 2006) and others (Paradis et al. 2005) have used the RNAi approach to suppress transcripts in bovine embryos and oocytes respectively. Recently, microinjection of dsRNA which interferes with genes that regulate cell polarity (Par3 and aPKC) in randomly selected blastomeres of cleavage-stage mouse embryos has enabled direction of defined cells to new positions and redirection of their fate in the preimplantation embryos (Plusa et al. 2005).

During oocyte maturation, meiotic resumption is characterised by germinal vesicle breakdown (GVBD), chromosomal condensation, progression to metaphase of the first meiosis release of the first polar body and then arrest at the metaphase of the second meiosis (MII) (Motlik & Kubelka 1990). The meiotic arrest (MII arrest) is maintained by the persistently high activity of cyclin B-p34^cdc2 kinase, also called maturation promotion factor (MPF) (Draetta & Beach 1988, Brizuela et al. 1989, Masui 1992, Fan & Sun 2004). MPF activity is necessary to maintain MII arrest in oocytes and the function of a multi-component complex, known as the cytostatic factor (CSF), which is required to sustain MPF activity (Hirao & Eppig 1997). CSF activity is the coordinated function of at least two proteins, MAP kinase (MAPK) and mos. The activation of MAPK mediates the activation of MPF, a key regulator of the M phase, and results in the induction of GVBD in xenopus (Gotoh & Nishida 1995, Kosako et al. 1996), mouse (Araki et al. 1996), bovine (Fissore et al. 1996) and porcine (Ohashi et al. 2003). Mos, the C-mos protooncogene product, is one of the central regulators of meiosis in vertebrate oocytes (Sagata 1996). Injection of mouse wild-type Mos RNA into bovine immature oocytes has induced a marked increase in the catalytic activity of MAPK and resulted in a considerable acceleration of GVBD, without affecting the ability of oocytes to progress to the MII stage (Fissore et al. 1996). However, so far, no clear evidence is available whether this kinase cascade in bovine is exclusively initiated by Mos or not. Inhibition of C-mos synthesis in mouse oocytes using RNAi has resulted in pathenogenetic activation (Wianny & Zernicka-Goetz 2000), as has been observed in mos^–/– knockout mouse.

Oct-4 belongs to the sub-group of octamer-binding proteins that bind by the POU domain to the promoter and enhancer regions of various genes with octamer sites (Ovitt & Schöler 1998). The Oct-4 gene is presumed to be involved in the maintenance of an undifferentiated state and also the determination or establishment of the germ line (Ovitt & Schöler 1998). Moreover, Oct-4 influences several genes expressed during early development, including Fgf-4, Rex-1, Sox-2, OPN, hCG, Utf-1 (Pesce & Schöler 2001) and INF (Ezashi et al. 2001). So far, the role and possible effect of C-mos and Oct-4 suppression in bovine oocytes and embryos have not yet been investigated. Therefore, here we investigated the effect of the suppression of C-mos and Oct-4 genes on the mRNA and protein expression during bovine embryogenesis. Moreover, biological effects of the suppression of these genes in oocytes and embryos will be assessed during in vitro development.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Synthesis of DNA template
Pairs of primers were designed according to bovine cDNA sequences found in GenBank (see Table 1 for details) using Primer Express Software v2.0 (Applied Biosystems, Foster City, CA, USA) to amplify C-mos and Oct-4 transcripts. These primers generated PCR amplicons corresponding to the coding sequence and the identity of the product was confirmed by sequencing. The first round of PCR amplification was performed using Taq DNA polymerase (Sigma). At first, the sample was heated at 95 °C for 5 min followed by 35 cycles of denaturing at 94 °C for 30 s, annealing at temperatures as indicated in Table 1 for 30 s and extension at 72 °C for 1 min. Following the last cycle, a 10-min elongation step at 72 °C was performed. The same PCR conditions have also been used for the second round of PCR amplification using a primer attached with T7 promoter (GTAATACGACTCACTATAGGG) to its 5'-end to generate two different templates for in vitro transcription to produce sense and antisense RNA strands (Table 1). These C-mos and Oct-4 specific templates were purified using the QIAquick PCR Purification Kit (Qiagen).

View larger version (40K): [in this window] [in a new window]   Figure 1 Two per cent agarose gel stained with ethidium bromide showing the bands of C-mos (435 bp) and Oct-4 (341 bp) dsRNA compared with DNA template used for in vitro transcription.

In vitro fertilization of oocytes
After maturation, COCs were transferred into four-well dishes containing 400 µl of fertilization medium (Fert-TALP) supplemented with 2 µg/ml of heparin (Sigma), 0.2 mM pyruvate (Sigma) and 25 µl/ml penicillinamine, hypotaurine, adrenaline (epinephrine) (PHE). A swim-up procedure has been applied to obtain motile sperm cells from frozen–thawed semen (Parrish et al. 1988). Briefly, frozen–thawed sperm cells were incubated in a tube containing 1.5 ml of sperm-TALP supplemented with 6 mg/ml BSA and 10 mM pyruvate for 50 min at 39 °C in a humidified incubator with 5% CO₂. After this time, the supernatant was recovered and centrifuged at 250 g for 10 min at room temperature to recover motile sperm cells as a pellet. In vitro fertilization was performed using a final concentration of 2 x 10⁶ sperm cells/ml in 400 µl fertilization drop containing a group of 50 COCs. Fertilization was initiated during co-incubation of spermatozoa and the matured oocytes for 20 h in the same incubator under the same temperature and atmospheric CO₂ content as used for maturation.

In vitro embryo culture
Following insemination, presumptive zygotes were stripped off from residual cumulus cells and attached spermatozoa by vortexing for 90 s in Charles Rosenkrans 1 (CR1) culture medium. After treatment, zygotes were washed once in fresh culture medium and cultured in groups of up to 50 zygotes in four-well dishes each containing 400 µl CR1 medium (Tesfaye et al. 2004) until day 8 after insemination. The CR1 medium is supplemented with 10% OCS, 20 µl/ml Eagle’s basal medium (BME) (amino acids) and 10 µl/ml Minimum essential medium (MEM) (non-essential amino acids) (Gibco BRL). Cleavage rate was assessed 48 h after insemination, while morula and blastocyst rate were determined at days 5 and 6–8 after insemination respectively. In vitro culture was also performed in a humidified atmosphere with 5% CO₂ at 39 °C.

Microinjection of dsRNA
In this study, two experiments were conducted to attain the objectives. In experiment 1, the effect of suppression of C-mos during oocyte maturation was assessed by microinjection of C-mos dsRNA at the immature oocyte stage. Oocytes used in this study were aspirated from cattle ovaries collected from a nearby slaughterhouse. Only good quality oocytes were selected for the experiment based on their morphological characteristics, mainly the intactness of the cumulus cells. Once the oocytes were selected, the cumulus cells were partially removed (Fig. 2A) by vortexing to avoid technical difficulties during microinjection of the dsRNA or water in the cytoplasm of the oocytes. Until used for microinjection, oocytes were held in tissue culture medium (TCM)-199 supplemented with 0.1% BSA (Sigma), 0.2 mM pyruvate and 50 µg/ml gentamycin sulphate (Sigma) in a humidified atmosphere with 5% CO₂ at 39 °C for 1–2 h. Prior to microinjection, immature oocytes were incubated for 20 min in TCM-199 supplemented with cytochalasin B at a final concentration of 8 µg/ml in order to reduce mechanical damage during injection (Paradis et al. 2005). Subsequently, in three experimental replicates, a total of 935 immature oocytes were divided into three groups: C-mos dsRNA-injected (group 1, n = 327), water-injected group (group 2, n = 303) and uninjected controls (group 3, n = 305).

View larger version (106K): [in this window] [in a new window]   Figure 2 Representative images of oocytes, whose cumulus cells are partially removed, before any treatment (A); some parthenogenetically developed oocytes after injection with C-mos dsRNA (B); those oocytes injected with water (C) and uninjected controls (D).

In both experiments, microinjection was performed on an inverted microscope (Leica DM-IRB) at 200x magnification. During microinjection a group of 50–60 immature oocytes or zygotes were placed in a 10 µl droplet of Hepes-buffered TCM 199 (H-TCM) under mineral oil and the dsRNA or water was placed in a 1 µl droplet near to the droplet containing the oocytes or zygotes. However, H-TCM medium was supplemented with cytochalasin B during the injection of immature oocytes to improve the survival rate of the oocytes after microinjection (Paradis et al. 2005). Injection was performed by aspiration of the dsRNA (10 µg/µl) or water into the injection capillary (Cook, Ireland, K-MPIP-3335-5). The injection volume of ~7 pl was estimated from the displacement of the meniscus of mineral oil in the capillary, which is 1 µm in diameter. The different treatment groups were injected one after the other, every time preparing a new dish with fresh medium. After microinjection, all groups of immature oocytes or zygotes were washed twice in CR1aa medium and set back into culture. Their survival rate was assessed 3–4 h after microinjection.

Oocytes and embryos collection
In order to assess the effect of sequence-specific dsRNA in oocytes and embryo on mRNA transcript abundance and protein expression, oocytes and embryos were collected at specific times after treatment for mRNA and protein analysis. In experiment 1, immature oocytes were cultured after microinjection with C-mos dsRNA or water until 48 hpi. While oocytes were cultured for 48 h after treatment to allow any parthenogenetic development, those used for transcriptional and protein expression analysis were collected at 24 h after micro-injection and subsequent maturation. In experiment 2, zygotes injected with Oct-4 dsRNA or water and uninjected controls were cultured in vitro until day 8 blastocyst stage to assess development. The resulting blastocysts from each treatment group were used for both transcription and protein analysis.

Prior to freezing for mRNA or protein expression analysis, all oocytes/embryos were washed twice with PBS (Sigma) and treated with acidic Tyrode pH 2.5–3 (Sigma) to dissolve the zona pellucida. The zona-free oocytes and embryos were further washed twice in drops of PBS and frozen in cryotubes containing minimal amounts of lysis buffer (0.8% Igepal (Sigma), 40 U/µl RNasin (Promega), 5 mM dithiothreitol (DTT) (Promega)). Samples for Western blot analysis were additionally treated with protease inhibitor (Sigma). Until used for RNA isolation or Western blotting, all frozen embryos were stored at –80 °C.

RNA isolation and reverse transcription
A total of three pools each containing 20 matured oocytes or ten blastocyst stage embryos were used for mRNA isolation using oligo (dT)₂₆ attached magnetic beads (Dynal, Oslo, Norway) following the manufacturer’s instruction. Briefly, oocytes or embryos in lysis buffer were mixed with 40 µl binding buffer (20 mM Tris–HCl with pH 7.5, 1 M LiCl, 2 mM EDTA with pH 8.0) and incubated at 70 °C for 5 min to obtain complete lysis of the embryo and release of RNA. Ten microlitres of oligo (dT)₂₅ magnetic bead suspension was added to the samples and incubated at room temperature for 30 min. The hybridised mRNA and oligo (dT) magnetic beads were washed three times with washing buffer (10 mM Tris–HCl with pH 7.5, 0.15 mM LiCl, 1 mM EDTA with pH 8.0). Finally, mRNA samples were eluted in 12 µl DEPC-treated water and reverse transcribed in 20 µl reaction volume containing 2.5 µM oligo (dT)12N (where N = G, A or C) primer, 4 µl of 5x first stand buffer (375 mM KCl, 15 mM MgCl₂, 250 mM Tris–HCl pH 8.3), 2.5 mM of each dNTP, 10 U RNase inhibitor (Promega) and 100 U of SuperScript II reverse transcriptase (Invitrogen). In terms of the order of adding reaction components, mRNA and oligo (dT) primer were mixed first, heated to 70 °C for 3 min, and placed on ice until the addition of the remaining reaction components. The reaction was incubated at 42 °C for 90 min and terminated by heat inactivation at 70 °C for 15 min.

Real-time quantitative PCR
Quantification of C-mos, Oct-4 and Histone 2a (H2a) as endogenous control mRNA in the oocytes/embryos of each treatment group was assessed by real-time quantitative PCR. Furthermore, independent maternal transcript growth differentiation factor 9 (Gdf-9) has been quantified in the three treatment groups of experiment 1 to assess the specificity of mRNA suppression by the C-mos dsRNA. Similarly, the E-cadherin transcript has been quantified in the three treatment groups of experiment 2 to investigate the specificity of mRNA degradation by Oct-4 dsRNA. Moreover, the fibroblast growth factor (Fgf-4), which is reported to be co-expressed with Oct-4 gene (Nichols et al. 1998), has been quantified in the treatment groups of experiment 2. The ABI Prism 7000 apparatus (Applied Biosystems) was used to perform the quantitative analysis using SYBR Green JumpStart Tag ReadyMix (Sigma) incorporation for dsDNA-specific fluorescent detection dye. Quantitative analyses of all studied transcripts were performed in comparison with H2a as an endogenous control (Robert et al. 2002) and were run in separate wells. The primer sequences were designed for PCR amplification according to the bovine cDNA sequences (Table 1) using Primer Express Software v2.0 (Applied Biosystems). Standard curves were generated for both target and endogenous control genes using serial dilution of plasmid DNA (10¹–10⁸ molecules). The PCRs were performed in 20 µl reaction volume containing 10 µl SYBR Green universal master mix (Sigma), optimal levels of forward and reverse primers and 2 µl of embryonic cDNA. During each PCR reaction, samples from the same cDNA source were run in duplicate to control the reproducibility of the results. A universal thermal cycling parameter (initial denaturation step at 95 °C for 10 min, 45 cycles of denaturation at 95 °C for 15 s and 60 °C for 60 s) were used to quantify each gene of interest. After the end of the last cycle, a dissociation curve was generated by starting the fluorescence acquisition at 60 °C and taking measurements every 7 s until the temperature reached 95 °C. Final quantitative analysis was done using the relative standard curve method as used in Tesfaye et al. (2004) and results are reported as the relative expression level compared to the calibrator cDNA after normalisation of the transcript amount to the endogenous control.

Western blot analysis
Groups of 120 matured oocytes and 50 embryos at day 7 blastocyst stage were used from each treatment group, which include C-mos or Oct-4 dsRNA-injected, water-injected and uninjected control. In order to assess the amount of protein available before treatment in immature oocytes, equal amount of immature oocytes were also used for protein analysis prior to treatment. The proteins were extracted from the oocytes or embryos in loading buffer (26% of Tris 1 M pH 6.8, 12% SDS, 20% 2-mercaptoethanol and 40% glycerol). Following boiling for 5 min, proteins were separated on a 14% SDS-PAGE gel. Proteins were then transferred onto nitrocellulose transfer membrane, pore size 0.45 µm (Protran, Schleicher & Schuell BioScience, Dassel, Germany) using Trans-Blot SD; semi-dry transfer cell (BioRad). The membrane was stained with Ponceau S to evaluate the transfer quality and blocked for 1 h in Tris-buffered saline (20 mM Tris pH 7.5, 150 mM NaCl) containing 0.05% Tween-20 (TBS-T) and 1% polyvinyl-pyrrolidone (PVP) (Sigma). The membrane was then incubated at 4 °C overnight with the anti-rabbit C-mos primary antibody (Stressgen, Victoria, Canada) or Oct-3/4 (N-19) goat polyclonal primary antibody (Santa Cruz Biotechnology, Heidelberg, Germany). The primary antibody was diluted 1:500 in TBS-T containing 0.1% PVP prior to use. After incubation with the primary antibody, the membrane was washed six times for 10 min in TBS-T and the hybridization with the secondary antibody was performed at room temperature for 1 h. The horseradish-peroxidase (HRP) conjugated donkey anti-rabbit secondary antibody (Amersham Bioscience) and donkey anti-goat IgG-HRP secondary antibody (Santa Cruz Biotechnology) were used as secondary antibodies for C-mos and Oct-4 protein detection, respectively. Both secondary antibodies were diluted 1:50 000 in TBS-T containing 0.1% PVP. The membrane was finally washed six times for 10 min in TBS-T. The peroxidase activity was detected using the ECL Plus Western Blotting Detection System (Amersham Bioscience) following the manufacturer’s instructions and visualized using Kodak BioMax XAR film (Kodak).

Differential cell staining of blastocysts
Differential staining of inner cell mass (ICM) and trophectoderm (TE) cells of bovine day 8 blastocysts from the three treatment groups was performed by incubating in freshly prepared permeabilising solution 1% Triton X-100 and 1 µg/µl propidium iodide in PBS containing 1 mg/ml BSA for 50 s. After washing twice in PBS–BSA medium, embryos were transferred into ethanol containing 0.03 µg/µl bisbenzimide (Hoechst 33258; Hoechst, Sigma), incubated for 4 min on an ice block and washed twice in PBS–BSA medium. Embryos were immediately mounted on glass slides and examined under fluorescence microscope to determine the number of ICM and TE cells.

Statistical analysis
The mRNA expression analysis for studied genes in all treatment groups was analysed based on the relative standard curve method. The relative expression data were analysed using the statistical analysis system (SAS) version 8.0 (SAS Institute Inc., Cary, NC, USA) software package. Differences in mean values between two or more experimental groups or developmental stages were tested using ANOVA followed by a multiple pair wise comparison using t-test. Differences of P<0.05 were considered to be significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Experiment 1: effect of C-mos dsRNA on oocyte maturation, mRNA and protein expression
Effect of C-mos dsRNA on in vitro maturation of oocytes
The survival rate of the oocytes due to physical injury during microinjection was determined 3–4 h after microinjection. As indicated in Table 2, no significant differences were observed in survival rate of immature oocytes injected with C-mos dsRNA and water (P>0.05). Compared to uninjected controls about 10% of injected oocytes did not survive the microinjection procedure. Sixty percent of oocytes injected with C-mos dsRNA showed extrusion of the first polar body after 24 h maturation, while only 50 and 44% of water injected and uninjected controls, respectively, showed first polar body extrusion as shown in Table 2. Moreover, about 2.5% of the oocytes injected with C-mos dsRNA developed parthenogenetically to the two-cell stage, while no parthenogenetic development was observed in the water-injected group and uninjected controls (Fig. 2).

View larger version (11K): [in this window] [in a new window]   Figure 3 Relative abundance of C-mos (A) and Gdf-9 (B) mRNA in in vitro produced bovine preimplantation stage embryos: immature oocyte (IM); mature oocyte (MO); 2-cell (2C); 4-cell (4C); 8-cell (8C); 16-cell (16C); morula (Mor); blastocyst (Bla). The relative abundance of mRNA levels represents the normalized quantity of mRNA compared to the calibrator (blastocyst stage) which is set to 100. Individual bars show the treatment mean ± S.D. Values with different superscripts (a, b, c, d) are significantly different (P0.05).

View larger version (13K): [in this window] [in a new window]   Figure 4 Relative abundance of C-mos (A), Gdf-9 (B) and H2a (C) transcripts in oocytes at 24 h post-injection of dsRNA or water and in uninjected controls. The relative abundance of mRNA levels represents the amount of mRNA compared to the calibrator (Uninj. control) which is set to 100; injected (inj). Individual bars show the mean ± S.D. Values with different superscripts (a, b) are significantly different (P0.05).

View larger version (43K): [in this window] [in a new window]   Figure 5 Western blot analysis for the presence of C-mos protein (39 kDa) in bovine oocytes following C-mos dsRNA and water injection in immature oocytes before any treatment and in uninjected controls. Uninjected (Uninj.); Injected (inj.).

View larger version (8K): [in this window] [in a new window]   Figure 6 Relative abundance of Oct-4 and Fgf-4 mRNA in in vitro bovine preimplantation stage embryos: immature oocyte (IM); mature oocyte (MO); 2-cell (2C); 4-cell (4C); 8-cell (8C); 16-cell (16C); morula (Mor); blastocyst (Bla). The relative abundance of mRNA levels represents the amount of mRNA compared to the calibrator (blastocyst stage) which is set to 100. Individual bars show the treatment mean ± S.D. Values with different superscripts (a, b, c, d, e) are significantly different (P0.05).

View larger version (16K): [in this window] [in a new window]   Figure 7 Relative abundance of Oct-4 (A), Fgf-4 (B), E-cadherin (C) and H2a (D) mRNA at blastocyst stage in the three treatment groups. The relative abundance of mRNA levels represents the amount of mRNA compared to the calibrator (uninjected control) which is set as 100. Individual bars show the mean ± S.D. Values with different superscripts (a, b) are significantly different (P0.05). Uninjected (Uninj.); Injected (inj.).

View larger version (38K): [in this window] [in a new window]   Figure 8 Western blot analysis results in the expression of Oct-4 protein (43 kDa) in bovine embryos injected with Oct-4 dsRNA and water, uninjected controls and in matured oocytes prior to fertilization. Uninjected (Uninj.); Injected (inj.).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

The microinjection procedure used to introduce the dsRNA in the present study is known to be advantageous over other techniques like electroporation (Grabarek et al. 2002) and transfection technique (Siddall et al. 2002) in controlling the amount of dsRNA to be introduced. In this method, physical injuries due to microinjection are inevitable. Consequently, in the present study, 10–15% of the oocytes and zygotes did not survive the microinjection procedure but this remains the same between the dsRNA- and water-injected groups. Therefore, variation in developmental capacity due to the microinjection procedure has been ruled out.

Previous studies have shown that the mechanism of RNAi is limited at the post-transcriptional level by degrading the sequence-specific mRNA or blocking the activity of ribosomal RNA (rRNA) (Svoboda 2004), which leads to a loss of function in mice (Svoboda et al. 2000, Wianny & Zernicka-Goetz 2000, Grabarek et al. 2002, Siddall et al. 2002). Our results also showed that the injection of dsRNA of oocyte- or zygote-specific transcripts induced sequence-specific mRNA degradation and prevented subsequent protein synthesis during development of preimplantation embryos. In the studies conducted in mouse, C-mos is known to play a role as an essential component of CSF, which is responsible for arresting the maturing oocytes at metaphase in the second meiotic division (Wianny & Zernicka-Goetz 2000). In this study, the injection of C-mos dsRNA at the immature oocyte stage resulted in 70% reduction in the amount of C-mos mRNA after maturation compared to the water-injected group and uninjected controls. This result is comparable with the results reported in mouse oocytes, where a suppression of 80% of C-mos mRNA was achieved by microinjection of C-mos dsRNA (Svoboda et al. 2000). Similar studies in mouse which targeted oocyte-specific maternal transcripts, namely Gdf-9 and Bmp-15, have shown the suppression of 89 and 78% in mRNA transcript abundance respectively (Gui & Joyce 2005). Moreover, up to a level of 90% suppression in transcript abundance has been attained for Plat (Svoboda et al. 2000), ITPRT (Xu et al. 2003) and BNC (Ma et al. 2002) genes in mouse oocytes. A complete degradation of Cyclin B1 mRNA has been achieved in the work of Lazar et al.(2004)) in rat oocytes treated with Cyclin B1 dsRNA. A recent report from our group (Nganvongpanit et al. 2006), and also from others (Paradis et al. 2005), have shown suppression of transcripts between 80 and 90% in bovine oocytes and embryos. The efficiency of targeted suppression of transcripts in mammalian oocytes or embryos seems to determine the extent of change in developmental phenotype. This variation in the efficiency of suppression of mRNA and protein synthesis and the expected developmental phenotype using dsRNA may be associated with the concentration of dsRNA introduced. This has been evidenced by Wianny & Zernicka-Goetz (2000), who have shown that 50% of the oocytes injected with 2 mg/ml C-mos dsRNA showed spontaneous activation while only 36% of the oocytes injected with 0.1 mg/ml C-mos dsRNA developed parthenogenetically to cleavage-stage embryos. Studies in C-mos^–/– knockout mouse have shown a reduced fertility because of the failure of mature eggs to arrest during meiosis (Colledge et al. 1994). The C-mos^–/– oocytes undergo GVBD and extrusion of both polar bodies, followed in some cases by progression into cleavage. In the present study, despite significant reduction in the transcript abundance and protein synthesis, the proportion of oocytes undergoing spontaneous activation after treatment with C-mos dsRNA was much lower compared to the studies in mouse (Wianny & Zernicka-Goetz 2000). In the present study, 60% of C-mos dsRNA-injected oocytes showed extrusion of the first polar body, of which 2.5% showed spontaneous activation and development to two- to four-cell stage. However, while only 44–50% of the oocytes showed first polar body extrusion in the water-injected group and uninjected controls, no spontaneous activation and parthenogenetic development has been observed in these treatment groups. The reason for the lower percentage of spontaneous activation in C-mos dsRNA-injected groups compared to comparable studies in the mouse cannot be explained at this level of the study.

The C-mos proto-oncogene is reported to be expressed at high levels in testes and ovaries, specifically in male and female germ cells (Kiessling & Cooper 1989). Quantitative expression analysis of C-mos and GDF-9 throughout the preimplantation stage showed a pattern similar to most maternal transcripts. High-level accumulation of C-mos transcript after 24 h in vitro maturation (IVM) in fully grown oocytes in this study is consistent with previous studies in various species including mouse (Goldman et al. 1987, Mutter & Wolgemuth 1987) and human oocytes (Pal et al. 1994). Similarly, growth stage dependent analysis of Mos synthesis in bovine oocytes showed a higher level of Mos product in oocytes as they reached the MII stage (between 22 and 26 h of IVM) compared to low synthesis during the first 4 h of IVM and no synthesis in ageing MII-stage oocytes at 44–48 h IVM (Wu et al. 1997). This expression pattern from our study and also from others may suggest the requirement of Mos expression during MII arrest and its possible role in MPF activity to maintain MII arrest. A significant level of C-mos and GDF-9 expression after fertilisation in this study may show the potential role of these transcripts in early stages of embryonic development. Our results have demonstrated that the injection of C-mos dsRNA leads to the specific degradation of the C-mos mRNA without affecting the expression of other genes (Gdf-9 and H2a). These results are consistent with the results obtained in mouse, where the injection of dsRNA directed towards C-mos mRNA resulted in the suppression of the targeted mRNA without affecting the untargeted transcript (Svoboda et al. 2000). Previous reports in bovine oocytes also showed that the suppression of Cyclin B1 had no effect on the expression of housekeeping gene (ß-actin) or Cyclin B2, as a member of the Cyclin B family (Paradis et al. 2005). Moreover, our results have demonstrated that degradation of mRNA has resulted in a consequent reduction of protein synthesis as is evidenced by Western blot analysis.

Bovine embryogenesis in the early preimplantation stages is supported by mRNA and protein transcribed from maternal and embryonic genome. Until the major round of embryonic transcription during the 8- to 16-cell stage in bovine embryos, development is largely dependent on the transcripts and protein formed by the oocyte (Memili & First 2000). Oct-4 is the earliest expressed transcription factor that is known to be crucial in murine preimplantation development (Okamoto et al. 1990, Rosner et al. 1990, Schöler et al. 1990). The mRNA and protein of Oct-4 have been found in murine oocytes and in the nuclei of subsequent cleavage stage embryos (Rosner et al. 1990, Schöler et al. 1990, Palmieri et al. 1994), while in the expanded murine blastocyst stage both mRNA and protein were predominantly found in the ICM (Palmieri et al. 1994, Pesce et al. 1998, Kirchof et al. 2000). However, even in fully expanded bovine and porcine blastocysts, both ICM and trophectoderm cells were found to be positive for Oct-4 protein (Kirchof et al. 2000). The quantitative expression profiling results throughout the preimplantation embryonic stages in the present study evidenced that Oct-4 is activated from both maternal and embryonic genome. Transcript abundance sharply increases after maturation and down-regulated until the four-cell stage. A higher level of Oct-4 transcript abundance at the matured oocyte stage was accompanied by the presence of a maternal protein product as confirmed by Western blot analysis (Fig. 8). This shows that Oct-4 is activated from both maternal and embryonic genome during bovine embryogenesis. The detectable amount of Oct-4 transcript was very low between the eight-cell and morula stages, after which it is upregulated at the blastocyst stage. Therefore, injection of Oct-4 dsRNA is targeting transcripts which are starting the minor embryonic activation at the two- to four-cell stages and in the major embryonic activation after the 16-cell stage. Consequently, injection of Oct-4 dsRNA at the zygote stage has resulted in a 72% reduction at the blastocyst stage compared with the uninjected controls. Despite slight variations in the relative abundance of Oct-4 transcript between the water-injected group and uninjected control group, differences are not significant. Similar studies in mouse have reported that suppression of about 90% has been achieved using sequence specific dsRNA (Svoboda et al. 2004).

Oct-4 as transcription factor protein is known to bind to DNA and activate or repress transcription of several genes expressed during early embryonic development (Shin et al. 2005). In the present study, suppression of Oct-4 transcript in bovine embryogenesis using dsRNA has resulted in co-suppression of Fgf-4 gene at a level of 70%, while the transcript remained unaffected in the water-injected group and uninjected controls. This is in agreement with the observation made in Oct-4^–/– mouse embryos, where Fgf-4 transcript abundance has been reduced (Nichols et al. 1998). Moreover, the expression of Fgf-4 transcript was found to be down-regulated after targeted suppression of Oct-4 using siRNA expression vector in mouse (Haraguchi et al. 2004). The Fgf-4 gene is an octamer-containing enhancer in its 3'noncoding region and has been demonstrated to respond to Oct-4 gene (Yuan et al. 1996, Ambrosetti et al. 1997, Daniels et al. 2000). Studies in mouse have shown that this gene is coexpressed with Oct-4 in the ICM and epiblast (Ma et al. 1992, Niswander & Martin 1992). Recently, the effect of down-regulation of Oct-4 transcript using dsRNA on the expression of other genes in mouse embryos has been investigated using annealing control primer technique (Shin et al. 2005) whereby, of the ten genes, eight (Atp6ap2, GK003, Ddb1, hRscp, Dppa1, Dpp3, Sap18, and Rent1) were down-regulated and two (Rps14 and ETIF2B) were up-regulated in Oct-4 dsRNA-injected blastocysts. The specificity of Oct-4 dsRNA on targeted mRNA has been investigated by quantitative expression analysis of another blastocyst transcript (E-cadherin) and a house-keeping gene (H2a).

We have demonstrated that degradation of Oct-4 mRNA resulted in consequent reduction in protein synthesis and in developmental aberrations. Oct-4 dsRNA injection has affected the cleavage rate of zygotes to develop to the two-cell stage. Even though the day 5 morula rate was lower in the Oct-4 dsRNA-injected group compared to the water-injected group and uninjected controls, these differences were not significant. In order to investigate the effect of Oct-4 suppression on the rate of embryo development, we have investigated the blastocyst rate from day 6 to day 8. Most of the blastocysts from Oct-4 dsRNA-injected groups appeared at days 7 and 8 while only few blastocysts were found at day 6 of development. However, a comparable developmental rate with respect to blastocysts rate between day 6 and day 8 has been observed in the water-injected group and uninjected controls. The overall blastocyst rate was lower in Oct-4 dsRNA-injected embryos compared to the water-injected group and uninjected controls but differences are not significant. While the Oct-4^–/– mouse showed a post-implantation lethality before egg cylinder formation, Oct-4-deficient mouse embryos developed normally up to blastocyst stage but the ICM were not pluripotent and divert to a trophoblast fate when placed in embryonic stem cell culture conditions (Nichols et al. 1998). Marked differences have been observed in Oct-4 mRNA and protein expression in murine and bovine species (Kirchof et al. 2000). As opposed to the study in mouse where Oct-4 expression is correlated with the undifferentiated cell types, suggesting that Oct-4 is a marker for pluritency and its expression is restricted to ICM (Ovitt & Schöler 1998), the Oct-4 protein was detected in both ICM and trophectoderm cells of murine and bovine expanded blastocysts, indicating that it may be the biological activity of the Oct-4, and not simply its presence, that correlates with the embryonic stem cell type (Kirchof et al. 2000).

In the present study, Oct-4 dsRNA-injected zygotes resulted in blastocysts of lower cell number compared to the water-injected group and uninjected controls. This was significantly evident in the number of ICM cells which were found to be reduced due to down-regulation of Oct-4 transcript. The optimal level of Oct-3/4 is reported to determine the fate of embryonic stem cells (Niwa et al. 2000), in which less than a twofold increase from the normal expression level causes differentiation into ectoderm and mesoderm, whereas a lower level leads to dedifferentiation into trophectoderm. However, due to absence of differences in the number of TE cells between the three groups, migration of cells to TE cells cannot be evidenced in the present study.

In conclusion, the present study has evidenced the use of sequence specific C-mos and Oct-4 dsRNA to induce RNAi in bovine oocytes and embryos, respectively, to suppress maternal or embryonic transcripts leading to subsequent reduction in functional protein expression and result in a distinct developmental phenotype with respect to oocyte maturation, the rate of embryonic development and cell number of the resulting blastocysts.

	Acknowledgements

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

	Footnotes

 
Received 11 November 2005
First decision 23 December 2005
Revised manuscript received 30 January 2006
Accepted 7 February 2006

	References

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