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Research Article |
National Research Laboratory,3 Department of Animal Sciences, Chungbuk National University, Chungbuk 361–763, South Korea
Laboratory of Reproductive Biology & Infertility,4 Samsung Cheil Hospital & Women's Healthcare Center, Sungkyunkwan University School of Medicine, Seoul 100–380, Korea
ABSTRACT
To investigate the role of nuclear encoded genes in mitochondrial function during oocyte maturation and early embryogenesis we examined the expression pattern and function of the cytochrome oxidase (Cox) subunits, Cox5a, 5b, and 6b1 during oocyte maturation and early embryo development. Transcription of Cox5a, 5b, or 6b1 was observed in oocytes and during early development; their expression levels were abundant in mature oocytes (MII) and zygotes (1C), and lowest at the 2-cell stage (2C), gradually increasing from 4-cell to blastocyst stage. Immunocytochemical studies revealed that COX5A, 5B, or 6B1 proteins were expressed in all blastomeres of the blastocyst. Silencing of mRNA expression by RNA interference (siRNA) did not inhibit oocyte maturation or developmental events up to the morula and blastocyst stages, but disrupted mitochondrial distribution. Significantly higher apoptosis and lower cell numbers were observed in siRNA-treated blastocysts. Real time RT-PCR revealed that silencing of Cox5a, 5b, or 6b1 did not alter mRNA levels of Bcl-xL (Bcl2l1), but increased transcription levels of proapoptotic genes, Bax and caspase 3 (Casp3). Furthermore, mRNA and protein levels of E-cadherin (CDH1) were decreased in siRNA microinjected blastocysts. These results suggest that gene expression of the Cox subunits, Cox5a, 5b, and 6b1 is not required for embryo developmental events up to the blastocyst stage. The loss of these genes leads to mitochondrial dysfunction that results in apoptosis of the blastocyst stage embryos.
apoptosis, embryo, oocyte development
INTRODUCTION
Oocyte mitochondria produce the energy required for the resumption of meiosis, fertilization, and development of the embryo [1]. Deficiencies in mitochondrial ATP production may be associated with impairment of oocyte fertilization or retarded embryonic development at later stages.
The biogenesis of competent mitochondria requires the joint expression of two genomes, mitochondrial and nuclear, because mitochondrial proteins are encoded in both of these genomes [2]. In eukaryotes, cytochrome oxidase (COX, encoded by the Cox gene) is a complex hetero-oligomer composed of subunits encoded by both nuclear and mitochondrial genomes. COX is the terminal enzyme of the respiratory chain and catalyzes the transfer of electrons from cytochrome c to oxygen, which is coupled to the translocation of protons across the inner membrane of the mitochondria [3, 4]. Although the role of COX in ATP production has been well characterized, much less information is available on regulatory mechanisms of COX subunit expression and their effects on mitochondrial function in early development.
In mammalian tissues, COX contains 13 subunits: 10 small subunits (4, 5a, 5b, 6a, 6b, 6c, 7a, 7b, 7c, and 8) encoded in the nucleus and synthesized in the cytoplasm [5], and 3 large catalytic subunits encoded by the mitochondria. The catalytic subunits are involved in redox-linked proton pumping [6]. The COX crystal structure shows that, of the 10 nuclear encoded subunits, subunits 5A (on the matrix side), 5B (inner membrane), and 6B1 (on the intermatrix side of the inner membrane) are involved in contacts that result in formation of dimers of the COX complex [7].
During oocyte maturation and early embryo development, various microenvironmental stresses such as Ca2+, reactive oxygen species, and ceramide can induce disruption of mitochondrial integrity that results in release of cytochrome c and activation of caspase 9 (CASP9) [8], followed by apoptosis and abnormal embryonic development. Despite clear evidence of regulation of mitochondrial respiration by nuclear encoded genes, little information is available on the expression and functional roles of nuclear encoded mitochondrial genes during early embryonic development. To examine the role of COX in oocyte maturation and embryogenesis, we first characterized mRNA and protein levels of Cox5a, 5b, and 6b1 in mouse oocytes and during early embryogenesis, using real time RT-PCR and immunocytochemistry. We then examined the possible role of these genes in oocyte maturation and preimplantation development using RNA interference analysis.
MATERIALS AND METHODS
Generation of Mouse Embryos
To obtain oocytes or fertilized embryos, 5-wk-old B6C3 F1 female mice (C57BL/6 female x C3H/He male) were induced to superovulate by intraperitoneal injections of 5 IU eCG (Sigma, St. Louis, MO), followed by 5 IU hCG (Sigma) 48 h later. Animals were treated according to institutional animal care and use guidelines. Germinal vesicle (GV)-stage oocytes were collected 45 h after the eCG injection and ovulated oocytes (MII-stage oocytes) were collected by puncturing the ampullae of the oviducts of superovulated female mice 16 h after hCG injection. One-cell (1C)-, 2-cell (2C)-, 4-cell (4C)-, morula (MO)-, and blastocyst (BL)-stage embryos were collected at 20, 40, 55, 82, and 96 h after hCG injection from the oviduct or uterus by puncturing or flushing, respectively. Cumulus cells were removed by brief exposure to medium containing 0.1% hyaluronidase (Sigma). The embryos were washed in Ca2+- and Mg2+-free PBS, and depending on the experiment, harvested embryos were either fixed with 4% formaldehyde (Sigma) for 20 min and stored at 4°C, or snap-frozen in liquid nitrogen and stored at –70°C until used.
Cox5a, 5b, and 6b1 siRNA Microinjection and In Vitro Culture
Germinal vesicle-stage oocytes or zygotes were obtained and denuded of cumulus cells. The GV oocyte collection medium was M2 (Sigma) supplemented with 300 µM dibutyryl cyclic adenosine monophosphate (dbcAMP, Sigma) to inhibit germinal vesicle breakdown (GVBD) during oocyte collection. After designing siRNAs that would silence mouse Cox5a, 5b, or 6b1 genes (siRNA ID No., 100292, 66454, and 78508, respectively; Ambion, Inc.), the chemically synthesized 21-nt sense and antisense RNAs were obtained and annealed commercially (Ambion). The siRNA was diluted with buffer (Ambion) to a final concentration of 100 µM and stored at –20°C. Approximately 10 pL of individual (Cox5a, 5b, or 6b1) siRNA was injected into the cytoplasm of the GV stage oocytes or zygotes using an Eppendorf microinjector system (Eppendorf). Additionally, three species of scrambled siRNA were also injected into zygotes. A previous study [9] reported that Oct3/4 (Pou5f1) siRNA (Ambion) significantly (P < 0.05) decreased Pou5f1 mRNA expression. Moreover, a preliminary study of individual siRNA injected blastocysts showed a similar target mRNA decrease (Fig. 1A) compared to the Pou5f1 siRNA-derived Pou5f1 mRNA expression in blastocyst stage embryos (development of siRNA-injected zygotes to the blastocysts) and significantly lower (P < 0.05 or 0.01) than Pou5f1 mRNA amounts in noninjected blastocysts (development of zygote to the blastocysts in in vitro culture condition). On the other hand, mRNA expression from the injection of siRNA dilution buffer alone was similar to that of the noninjected group, which did not reduce Cox5a, 5b, or 6b1 or Pou5f1 mRNA expression (Fig. 1B). Therefore, in the present study, Pou5f1 siRNA-injected embryos were used for a positive control, and negative control groups were injected with an equal volume of buffer and considered as the control in the present study. Individual siRNA-microinjected GV-stage oocytes and zygotes were then cultured in M16 (Sigma) medium supplemented with 0.4% BSA at 37°C in a humidified atmosphere of 5% CO2 and 95% air to examine rates of maturation and development to the blastocyst stages.
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Real-Time RT-PCR
Frozen-thawed embryos were used for analysis of mRNA quantities. Messenger RNA was extracted using the Dynabeads mRNA Direct Kit (Dynal Asa) according to the manufacturer's instructions. For the external reference, rabbit globin mRNA (Sigma) [10] was added (0.1 pg per oocyte or embryo) before mRNA extraction. First, standard cDNA synthesis was achieved by reverse transcription of the RNA by using the Oligo (dT)12–18 primer and the Superscript reverse transcriptase enzyme (Invitrogen Co.).
Real time RT-PCR was performed using 12 primer sets shown in Table 1. The threshold cycle (Ct) value represents the cycle number at which sample fluorescence is statistically higher than background. The reactions were conducted according to the protocol of the DyNAmo SYBR green qPCR kit that contains modified Tbr DNA polymerase, SYBR Green, optimized PCR buffer, 5 mM MgCl, and a dNTP mix that includes dUTP (Finnzyme). The PCR protocol involved a denaturation program (95°C for 10 min) followed by an amplification and quantification program repeated 40 times (95°C for 15 sec, 55/60°C for 15 sec, 72°C for 15 sec with a single fluorescence measurement), a melting curve program (65–95°C, with a heating rate of 0.2°C per second and continuous fluorescence measurement) and finally a cooling step to 4°C. Fluorescence data were acquired after the extension step during PCR reactions that contained SYBR Green. The PCR products were then analyzed by generating a melting curve. Because the melting curve of a product is sequence-specific, it can be used to distinguish between nonspecific and specific PCR products. To use the mathematical model, it is necessary to determine the crossing points for each transcript. The crossing point is defined as the point at which the fluorescence rises appreciably above the background fluorescence. The relative quantification of gene expression was analyzed by the 2-ddCt method [11]. In all experiments, histone H2a (H2a) mRNA was used as an internal standard, and rabbit globin mRNA was used as an external reference in the analyses of relative mRNA expression levels of Cox subunits in various developmental stages of oocytes and embryos (Fig. 2C). The sizes of the RT-PCR products were confirmed by gel electrophoresis on a standard 2% agarose gel, stained with ethidium bromide, and visualized by exposure to ultraviolet light. The PCR products were sequenced (Applied Biosystems, Model 373A Automated Sequencer, Oklahoma State University Recombinant DNA/Protein Resource Facility) after purification and sequences confirmed by BLAST [12].
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Immunofluorescence Staining
To determine the expression and distribution of COX5A, 5B, 6B1, and E-cadherin (CDH1) protein, mouse embryos were fixed with 4% formaldehyde for 20 min and permeabilized with 0.2% Triton X-100 for 10 min. The embryos were then incubated with mouse COX5A, 5B, or 6B1 (A21363, A21349, and A21366, respectively, Molecular Probes, Inc.) and CDH1 (U3254, Sigma) monoclonal antibodies for 1 h, and then with FITC-labeled secondary antibodies (Sigma). Propidium iodide (PI, Sigma) was used to stain the nuclei. Slides were examined using laser-scanning confocal microscopy, which was performed using a Leica DM IRB equipped with akrypton-argon ion laser for the simultaneous excitation of fluorescence for proteins and PI for DNA.
Mitochondrial Staining and Image Analysis
To localize mitochondria, mouse blastocysts were fixed with 4% formaldehyde for 20 min, washed two times with PBS containing 0.1% PVA, and incubated with MitoTracker dye (M-7514, Molecular Probes, Inc.) for 30 min to stain the mitochondria. The nuclei were stained with PI. The phase-contrast images of individual samples were digitized using laser-scanning confocal microscopy (Leica Laser Technik GmbH) and MetaMorph software (Version 6.1; Universal Imaging Corporation) and were saved.
TUNEL Assays
Blastocysts were washed three times in PBS (pH 7.4) containing polyvinylpyrrolidone (PVP, 1 mg/ml). This was followed by fixation in 3.7% formaldehyde in PBS for 1 h at room temperature (RT). After fixation, the embryos were washed in PBS/PVP and permeabilized by incubation in 0.3% Triton X-100 for 1 h at RT. The embryos were then washed twice in PBS/PVP and incubated with fluorescein-conjugated dUTP and the terminal deoxynucleotidyl transferase enzyme (in situ Cell Death Detection Kit, Roche) in the dark for 1 h at 37°C. After being counterstained with 40 µg/ml PI and 50 µg/ml RNaseA for 1 h at 37°C to label all nuclei, embryos were washed in PBS/PVP, mounted with slight coverslip compression, and examined under a confocal microscope.
For other experiments, embryos were fixed in 3.7% formaldehyde in PBS for 1 h at RT and stained with 40 µg/ml PI for 1 h at 37°C to label all nuclei, and then total cell numbers were counted under a fluorescence microscope at 400x magnification (Olympus) [13, 14].
Transmission Electron Microscopy
Blastocysts developed from Cox5b siRNA or buffer solution (control) microinjected zygotes were fixed in 2% glutaraldehyde in phosphate-buffered saline. The specimens were then postfixed in 2% OsO4 for 1h, dehydrated in a graded ethanol series (50%, 70%, 80%, 95%, and 100%), and embedded in epoxy resin. Ultrathin sections were cut with a diamond knife and poststained first with 1% uranyl acetate in 30% ethanol and then with Reynolds lead citrate. A transmission electron microscope (Hitachi 600, Hitachi Ltd.) was used to examine mitochondria in mouse embryos. Negative film was digitalized with a scanner (Epson GT-9000) and archived on an erasable magnetic diskette.
Statistical Analysis
The general linear models (GLM) procedure in the SAS program [15] was used to analyze the data from all experiments. Significant differences were determined using the Tukey multiple range test [16] and P < 0.05 was considered significant.
RESULTS
Expression of Cox5a, 5b, and 6b1 in Mouse Oocytes and Embryos
The relative abundance of Cox5a, 5b, and 6b1 transcripts was measured by real-time RT-PCR using the 2-ddCt method. Ten embryos per treatment group were quantified three times, and four repetitions were completed (Fig. 2). To normalize the RT-PCR reaction efficiency, and to quantify Cox subunit mRNA amounts, H2a was used as an internal standard (Fig. 2A). Samples were also normalized by an external reference of rabbit globin mRNA. After normalization with H2a mRNA, the mRNA expression of Cox5a, 5b, and 6b1 showed similar patterns throughout development (Fig. 2B), in that the expression levels were abundant in MII oocytes and in 1C zygotes, and the lowest at the 2C stage, gradually increasing from the 4C to the BL stage. Expression of mRNA was dependent on the number of blastomeres; it increased only gradually with development. When normalized to the external reference, Cox5a, 5b, or 6b1 mRNA expression increased more sharply in MO- and BL-stage embryos (Fig. 2C). However, COX5A, 5B, or 6B1 proteins were detected only in the blastocyst (Fig. 3) by immunofluorescent staining using individual primary antibody (Molecular Probes), and FITC-labeled anti-mouse IgG (Sigma) secondary antibody.
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Effect of Cox5a, 5b, or 6b1 siRNA on Mouse Oocyte Maturation and Embryo Development
GV-stage oocytes microinjected with Cox5a, 5b, or 6b1 siRNA showed similar percentages of GV-stage arrests (GVA, 18.6% ± 7.0%, 27.1% ± 7.0%, and 25.9% ± 7.0% vs. 16.4% ± 7.0% control; Fig. 4A) and first polar body extrusion (MII, 68.5% ± 4.8%, 60.8% ± 4.8%, and 64.4% ± 4.8% vs. 71.5% ± 4.8% control) compared to controls. Additionally, development of siRNA treated zygotes to the cleavage (post-hCG 40–44 h, 97.8% ± 8.3%, 94.1% ± 5.4%, and 97.8% ± 6.5% vs. 93.9% ± 4.7%, control; Fig. 4B), morula (post-hCG 82–84 h, 90.6% ± 10.8%, 81.6% ± 9.1%, and 92.8% ± 8.9% vs. 83.7% ± 7.9%) and blastocyst (post-hCG 94–96 h, 71.1% ± 8.4%, 76.8% ± 5.8%, and 73.9% ± 8.4% vs. 81.7% ± 7.1%) stages was not different from controls. Similarly, injecting scrambled compounds of siRNA did not affect development of zygotes to the blastocysts (73.1% ± 6.4%).
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Total cell numbers in blastocysts developed in vitro from individual siRNA injected zygotes was significantly (P < 0.05) less than controls (36.2% ± 4.1%, 33.4% ± 3.1%, and 33.0% ± 3.5% vs. 47.5% ± 2.5%, control, Fig. 5). DNA fragments resulting from the apoptotic nicking of genomic DNA were measured in individual embryos using the TUNEL assay. Each microinjected siRNA increased apoptosis in blastocyst stage embryos (11.8% ± 0.8%, 10.5% ± 0.9%, and 9.8% ± 0.8% vs. 2.5% ± 0.2%, control, P < 0.01).
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Effect of Cox5a, 5b, or 6b1 siRNA on Target Gene Expression
The relative amounts of Cox5a, 5b, and 6b1 mRNA expression in individual siRNA injected in vitro cultured oocytes or embryos of various stages were measured by real-time RT-PCR. To normalize the RT-PCR reaction efficiency, mouse H2a was used as an internal standard. Ten embryos per treatment group and each experiment was repeated four times, with three replicates. Injection of Cox5a (Fig. 6A), 5b (Fig. 6B), or 6b1 (Fig. 6C) siRNA into GV-stage oocytes decreased expression of the target mRNA, but did not affect the expression of mRNAs for the other subunits in mature oocytes (MII, Fig. 6). Similarly, each siRNA injection into zygotes specifically reduced target mRNA expression at the 2C (P < 0.01, 0.001, and 0.01; Cox5a, 5b, and 6b1, respectively), MO (P < 0.001, 0.01, and 0.05; Cox5a, 5b, and 6b1, respectively), and BL (P < 0.05, 0.001, and 0.01; Cox5a, 5b, and 6b1, respectively) stages (P < 0.05, Fig. 6.), and when scrambled siRNA compounds were injected into the zygote, this also significantly decreased Cox5a, 5b, or 6b1 mRNA expression in blastocysts (Fig 6, D). Thus, the injection of individual siRNA into mouse zygotes knocked down expression of the corresponding mRNA during subsequent development in vitro. In addition, immunofluorescent staining showed that the siRNA injection reduced COX5A, 5B, or 6B1 protein levels in the blastocyst stage (Fig. 7).
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Effect of Cox5a, 5b, or 6b1 siRNA on Mitochondria
Mitochondrial DNA type 9 (mt-DNA9) and mitochondrial genome (mt-Genome) mRNA expression was measured in blastocysts by real-time PCR (Fig. 8). The expression level of mt-DNA9, as well as overall levels of mitochondrial mRNAs, was not different following injection of siRNA for Cox5a, 5b, or 6b1. However, it is evident that the number of mitochondria in siRNA treated blastocysts was greatly reduced and appears morphologically abnormal (Fig. 9).
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Ten images per control or siRNA treatment group were calibrated to µm units and analyzed using MetaMorph software. Background fluorescence was efficiently subtracted with the built-in "Flatten background" tool. Mitochondrial body parameters (area) were obtained applying the "Auto-Trace" function and Integrated Morphometry Analysis. Mitochondrial area was significantly decreased in Cox5a (P < 0.01, Fig. 10), 5b (P < 0.01), and 6b1 (P < 0.005) siRNA-injected blastocysts compared with buffer solution-injected (control) embryos.
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Electron micrographs showed morphological differences of mitochondria in blastocysts from both buffer-injected and Cox5b siRNA-injected (Fig. 11). Whereas oval or pleomorphic shapes of mitochondria are observed in blastocysts following buffer injection, abnormal or absent mitochondria are observed in blastocysts following Cox5b siRNA injection.
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Effect of Cox5a, 5b, or 6b1 siRNA on Nontarget Genes
The genes Pou5f1 and Cdh1 are well known for their function in early embryo development. Cdh1 plays key roles in the differentiation of the trophectoderm and supports further embryonic development [17]. Pou5f1, a prime candidate for an early developmental control gene, encodes a transcription factor required for embryo development [18, 19]. After Cox5a, 5b, or 6b1 siRNA microinjection into zygotes, transcription of Pou5f1 and Cdh1 was unchanged at the morula stage (Fig. 12A), but Cdh1 expression was significantly reduced in the Cox5a, 5b, or 6b1 siRNA-treated blastocysts (P < 0.05, 0.001, and 0.05; Cox5a, 5b, or 6b1 siRNA-injected embryos, respectively; Fig. 12B) compared to the controls (buffer solution injected). Moreover, immunofluorescence staining confirmed that Cox5a, 5b, or 6b1 siRNA treatments reduced CDH1 protein levels in blastocysts (Fig. 13).
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Effect of Cox5a, 5b, or 6b1 siRNA on Apoptotic-Related Genes
Apoptosis, a type of programmed cell death, is a common feature of mammalian development [20]. Two major families of apoptotic genes, the Bcl2 family and the caspase family, were expressed in the preimplantation stage of embryo development [21, 22]. Analysis of apoptotic-related genes' mRNA expression, namely Bcl-xL (Bcl2l1), Bax, and Caspase 3 (Casp3), in blastocyst following individual siRNA microinjection was performed three times with three replicates by real-time PCR (Fig. 14). Mouse H2a was used as an internal standard. After siRNA microinjection, antiapoptotic gene Bcl2l1 mRNA expression showed no statistical difference compared with a buffer solution-injected group; however, mRNA expression of prapoptotic ngenes Bax and Casp3 were increased in blastocysts (P < 0.05).
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DISCUSSION
Cytochrome c oxidase (COX) is a component of the mitochondrial respiratory chain and it oxidizes cytochrome c and transfers electrons to molecular oxygen to form water. Among the 13 COX subunits, COX5A, 5B, and 6B1 are notable because they are encoded by nuclear genes, but little information characterizing regulation and coordination of their expression, or the effects of these proteins on expression of other cell components, is available. In the present study, we explored the expression patterns of Cox5a, 5b, and 6b1, and their possible role in mouse oocyte maturation and preimplantation development. We first determined that Cox5a, 5b, and 6b1 mRNAs were present in mouse preimplantation embryos using quantitative real time RT-PCR and immunocytochemistry. After normalization of real time RT-PCR results against H2a expression [23, 24], we found that Cox5a, 5b, and 6b1 transcripts were highly expressed in oocytes and zygotes, whereas expression levels were significantly reduced at the 2-cell stage. The expression level of each transcript gradually increased in the morula and blastocyst stages. Considering the onset of zygotic genome activation, Cox5a, 5b, or 6b1 transcripts detected in 1-cell zygotes and 2-cell embryos represent maternal expression; and after the 4-cell stage, they represent transcription from the embryonic genome.
In the present study, we observed protein synthesis of COX primarily in the blastocyst stage, but not in the cleavage or morula stage. Oocyte mitochondria and preimplantation stage embryos have been shown to have low levels of metabolic activity, low respiratory rates and O2 consumption, and limited glucose metabolism [25]; mitochondria at these stages generate ATP at relatively low levels when compared with the morula or blastocyst stages [26]. Thus, protein expression of COX5A, 5B, or 6B1 may not be needed for the energy requirements of the embryo until the blastocyst stage. Collectively, COX5A, 5B, and 6B1 protein levels are readily detectable in the blastocyst stage, consistent with increased mitochondrial respiration at this stage.
We observed that specific silencing of mRNA and protein expression of Cox5a, 5b, or 6b1 using double-strand RNA did not influence oocyte maturation and early embryo development, but significantly increased apoptosis at the blastocyst stage. Furthermore, introducing siRNA disrupts mitochondrial function without changing mRNA expression of mt-Genome or mt-DNA9. These results suggest that Cox5a, 5b, or 6b1 did not influence either mitochondrial gene function or differentiation processes during early embryonic development. However, loss of these subunits may lead to disruptions in regulation of ion transport, which probably results in apoptosis.
The molecular mechanism by which gene silencing of Cox induces apoptosis is not clear. Apoptosis results from changes in mitochondrial integrity caused by various effectors, such as Ca2+, ROS, or production of Bax, that lead to the release of cytochrome c and activation of the caspase cascade [27]. In the present study, we found that gene silencing of Cox5a, 5b, or 6b1 enhances Bax and Casp3 gene expression without changing the expression level of Bcl2l1. This observation supports the hypotheses that nuclear encoded Cox genes regulate cytochrome c transport. Reduction of COX5A, 5B, or 6B1 protein levels may also lead to release of cytochrome c and changes in the mitochondrial membrane potential, which then leads to apoptosis. Collectively, our results suggest that silencing of the Cox5a, 5b, or 6b1 genes interrupts mitochondrial function and results in apoptosis at the time of blastocyst formation.
Interestingly, we observed that silencing of Cox5a, 5b, or 6b1 reduced mRNA and protein expression of Cdh1. In mouse preimplantation embryos, CDH1-mediated cell adhesion initiates compaction at the 8-cell stage [28, 29]. Subsequently, epithelial tight junction proteins are expressed and assembled at the apicolateral contact region when blastocoel cavitation begins. Cell adhesion events also coordinate the cellular allocation and spatial segregation of the inner cell mass of the blastocyst, and the maintenance of epithelial trophectoderm and nonepithelial phenotypes during early morphogenesis [30]. In the present study, silencing of Cox genes did not reduce mRNA or protein levels of CDH1 at the morula stage, but both mRNA and protein were reduced at the blastocyst stage. These results indicate that Cox5a, 5b, and 6b1 are not directly involved in Cdh1 expression, but that the associated apoptosis may disrupt Cdh1 expression. This hypothesis is consistent with experiments in which application of drugs that target the mitochondrial death pathway resulted in loss of adhesion and correlated with degradation of CDH1 [31].
In conclusion, our study suggests that gene expression of the Cox subunits, Cox5a, 5b, or 6b1, is not required for embryo developmental events up to the blastocyst stage. The loss of these genes leads to mitochondrial dysfunction that results in apoptosis of the blastocyst-stage embryos.
FOOTNOTES
2 Correspondence: Nam-Hyung Kim, Department of Animal Sciences, Chungbuk National University, Gaesin-dong, Cheongju, Chungbuk, 361–763, South Korea. FAX: 82 43 272 8853; nhkim{at}chungbuk.ac.kr 
1 Supported by the Ministry of Science and Technology (NRL), the Ministry of Agriculture and Forestry (Bio-Organ Production Project), and Research Center for Bioresource and Health in Chungbuk National University.
Received: 15 July 2005.
First decision: 12 August 2005.
Accepted: 15 November 2005.
REFERENCES
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  Y. Kameyama, F. Filion, J. G. Yoo, and L. C Smith Characterization of mitochondrial replication and transcription control during rat early development in vivo and in vitro Reproduction, February 1, 2007; 133(2): 423 - 432. [Abstract] [Full Text] [PDF]
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