| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Embryo |
Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba 277-8562, Japan
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
early development, embryo, fertilization, gene expression, gene regulation, nascent mRNA, mouse
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Zygotic gene activation (ZGA) occurs in two phases: an initial low level of activation followed by a larger second burst of activation. Initial activation occurs during the late one-cell stage, at which point the chromatin structure is not repressive and genes are transcribed independently of enhancers. The burst of ZGA occurs during the late two-cell stage. After DNA replication at the two-cell stage, repressive chromatin structures are established, and enhancers are necessary for transcription. The chromatin structure thus dramatically changes during development between fertilization and the late two-cell stage, which implies similarly dramatic changes in the profiles of the genes transcribed during these developmental stages [2, 5, 7, 8].
Little is known about the precise profiles of the genes transcribed in zygotes. Although a number of reports have shown changes in the amounts of various species of mRNA during preimplantation development [9–12], and very recently, global expression profiles have been examined by using microarrays [13, 14], the mRNA examined in these studies was that which had accumulated in the embryos. Thus, these profiles may not always reflect the profiles of the genes being actively transcribed at the time of analysis [15]. When the profiles of transcribed genes are changing rapidly and dramatically, the limits of these approaches are even more salient. Furthermore, the accumulated mRNA includes not only the mRNA transcribed from the zygotic genome but also the maternally derived mRNA stored in the cytoplasm of the early preimplantation embryo. Thus, analysis of the total mRNA pool does not seem to provide useful information for the investigation of the regulation of zygotic gene expression, although it is useful for studies of cellular functions. This lack of information about zygotic transcription has hampered the investigation of the regulation of zygotic gene activation.
To overcome these problems, we developed a novel method for isolating nascent mRNAs synthesized in a short time, from the large pool of preexisting mRNA. In this method, the embryos are loaded with 5-bromouridine 5'-triphosphate (BrUTP) by electrical permeabilization. BrUTP is a halogenated RNA precursor that has been shown to be a good substrate for RNA polymerase II in mouse preimplantation embryos [5, 8, 16]. After a short incubation period, the total mRNA is extracted from the embryos and BrU-labeled mRNA is isolated by immunoprecipitation with an antibody to BrdU that recognizes mRNA with incorporated BrU [5, 16]. In the current study, we first optimized the conditions for electrical permeabilization and immunoprecipitation. Using eIF-1A as an indicator gene, we then verified that this method could isolate nascent mRNAs from the preexisting mRNA pool. Finally, we examined the abundance of certain transcripts in the nascent mRNA fraction and in the total mRNA pool. We found that changes in the amount of total mRNA do not always reflect similar changes in the amount of newly transcribed mRNA. This method will thus be useful for the analysis of gene expression profiles during embryonic development.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Several media were used for handling the oocytes and embryos. Whitten medium contained 109.51 mM NaCl, 4.78 mM KCl, 1.19 mM KH2PO4, 1.19 mM MgSO4, 22.62 mM NaHCO3, 5.55 mM D-glucose, 0.31 mM sodium pyruvate, 1.49 mM calcium lactate, 0.075 mg/ml sodium penicillin-G, 0.05 mg/ml streptomycin sulfate, and 3 mg/ml BSA [17].
Glucose-free CZB medium contained 81.62 mM NaCl, 4.83 mM KCl, 1.18 mM KH2PO4, 1.18 mM MgSO4, 25.12 mM NaHCO3, 1.7 mM CaCl2, 6.9 mM taurine, 0.11 mM EDTA· 2Na, 0.27 mM sodium pyruvate, 31.3 mM DL-lactic acid (sodium salt, 60% syrup), 0.075 mg/ml penicillin-G, 0.05 mg/ml streptomycin, and 3 mg/ml BSA [18].
The medium used for electrical permeabilization was composed of 0.25 M D-glucose, 100 µM CaCl20.2H2O, 100 µM MgSO4, 0.1% PVP, and 10 mM BrUTP (Sigma, St. Louis, MO).
Collection and Culture of Oocytes and Embryos
Female mice, 21–23 days of age (BDF1; SLC, Shizuoka, Japan), were superovulated with 5 IU of equine chorionic gonadotropin (eCG; Sankyo Co., Ltd., Tokyo, Japan) and, 48 h later, with 5 IU of human chorionic gonadotropin (hCG; Sankyo Co.). The oocytes were collected in Whitten medium from the ampullae of the oviduct at 15–16 h after hCG injection. Sperm were obtained in Whitten medium from the cauda epididymis of mature male ICR mouse (SLC). The oocytes were inseminated with capacitated sperm that had been incubated for 2 h at 38°C. The embryos were washed with glucose-free CZB medium 2.5 h after insemination and then cultured in a humidified 5% CO2/95% air atmosphere at 38°C. All procedures described here were reviewed and approved by the University of Tokyo Institutional Animal Care and Use Committee and were performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals.
Examination of mRNA Abundances Using Semiquantitative, Real-Time, Florescence-Monitored Reverse Transcription-PCR
The abundances of selected mRNA transcripts were measured using reverse transcription-PCR (RT-PCR). Total RNA was isolated from the unfertilized oocytes and embryos using ISOGEN (Nippon Gene, Tokyo, Japan) and reverse transcribed in a 20-µl reaction mixture containing 5 U of ReverScript II (Wako, Osaka, Japan) and 0.5 µg of oligo(dT)12–18 primer (Invitrogen Corp., Carlsbad, CA) at 42°C for 1 h and 51°C for 30 min. The template mRNA was digested with 60 U of RNase H (TaKaRa, Shiga, Japan) at 37°C for 20 min.
The amounts of selected cDNAs were determined by real-time PCR using Smart Cycler System (TaKaRa) as described previously [19]. Briefly, the reaction mixture consisted of cDNA, 0.2 µM of each primer, 300 µM dNTP, 3 mM MgCl2, and 0.05 U/µl TaKaRa Ex Taq DNA polymerase (TaKaRa). The sequences of the PCR primers used are shown in Table 1. The double-strand DNA dye SYBR Green I (BioWhittaker Molecular Applications, Rockland) was added to permit real-time monitoring of the PCR products. PCR was performed using 35 cycles, consisting of denaturation at 95°C for 15 sec, annealing at the temperatures shown in Table 1 for 15 sec, extension at 72°C for 20 sec, and measurement of fluorescence at the temperatures shown in Table 1 for 6 sec. The annealing temperature was set at that temperature that yielded a single band upon electrophoresis of the products of real-time PCR
|
The relative amounts of each cDNA in the samples were determined using a cDNA standard curve obtained by the amplification of a 10-fold dilution series of a cDNA of known copy number that had been prepared by PCR. For the determination of the copy number of the cDNAs, the PCR products for the cDNA standard curve were purified using the PCR Clean-Up System (Promega, Madison, WI), the concentration was measured on a spectrophotometer, and the number of copies was calculated.
Preparation of BrU-Labeled Control mRNA and Unlabeled Blocking mRNA
Rabbit 
-globin mRNA (Sigma) was reverse transcribed with a DraI-anchored T30 oligo-dT primer and subcloned into the pGEM-T Easy Vector (Promega). BrU-labeled -globin mRNA was synthesized in vitro from the linearized construct using SP6 RNA polymerase (Roche, Mannheim, Germany) with BrUTP in the nucleotide mix.
A plasmid containing the coding region of ovine interferon-
gene in the pGEM-T Easy Vector was provided by Dr. K. Imakawa and Dr. H. Nojima. The linearized plasmid was used as a template for the synthesis of unlabeled mRNA that was used as a blocking agent in the immunoprecipitation experiments.
Isolation of Nascent mRNA by Immunoprecipitation Following BrUTP-Labeling
Embryos were thoroughly washed twice in electrical permeabilization (EP) medium. They were then transferred into the fusion chamber between the electrodes, overlaid with a 100-µl droplet of EP medium containing 10 mM BrUTP, and permeabilized using two 13- to 500-µsec electrical pulses at 1500 V/mm output voltage, with a 2-min interval between pulses. Two minutes after permeabilization, the excess BrUTP was washed from the cells, and they were cultured in CZB medium at 38°C in a 5% CO2 atmosphere until use. The embryos were divided into two groups: one was used for the isolation of nascent mRNA incorporating BrU and the other was used to verify the efficiency of BrUTP loading by immunostaining to confirm that BrU had been successfully incorporated in almost all of the embryos.
Total RNA was prepared from the BrUTP-loaded embryos using ISOGEN (Nippon Gene) as described above. The nascent mRNA with incorporated, BrU was isolated from the total RNA by immunoprecipitation using 20 µl protein G-Sepharose beads (Sigma) bound to mouse monoclonal anti-BrdU antibody (Roche, Indianapolis, IN) that also recognizes BrU. One hundred embryos were used for each immunoprecipitation.
The Sepharose beads were prepared by washing three times in PBS containing 0.1% polyvinylpyrrolidone (PBS/PVP) and resuspending the final pellet (10-µl aliquots) in 20 µl of PBS/PVP containing 2 µg of anti-BrdU antibody; 500 ng of unlabeled interferon- mRNA was added. The mixture was incubated at room temperature for 1 h with gentle rocking, and the beads were washed three times and resuspended in 0.2 ml of PBS/ PVP.
The total RNA samples isolated from the embryos were denatured by heating at 80°C for 10 min and then added to the beads. The mixtures were incubated at room temperature for 1 h with gentle shaking, and the beads were then washed five times with 0.2 ml of PBS/PVP containing 20 U of RNasin (Promega). The final pellet was resuspended in 10 µl of PBS and heated at 80°C for 10 min. The beads were pelleted again, and 10 µl of the supernatant was used for quantification of the mRNA by real-time RT-PCR. During the procedure of RNA precipitation, 500 ng of unlabeled interferon- mRNA was included as a blocking agent. All the solutions were made in diethyl pyrocarbonate-treated water. To control for the background, embryos were subjected to the same procedure as described above except for electrical permeabilization, and the value obtained from these embryos was subtracted.
Immunofluorescent Detection of mRNA Incorporating BrUTP in Cells
Immunofluorescence microscopy was performed to detect the nascent mRNA incorporating BrU within the embryos. One hour after BrUTP loading, the embryos were fixed for 1 h with 3.7% paraformaldehyde in PBS/PVP. After a wash with PBS/PVP, the embryos were incubated with the anti-BrdU antibody (1:50 dilution) for 60 min and then with a fluorescein (FITC)-conjugated antimouse antibody (1:500 dilution; Jackson ImmunoResearch Laboratories, West Grove). The cells were mounted on glass slides with VectaShield (Vector Laboratories, Burlingame, CA) and observed with a confocal laser-scanning microscope (LSM510; Carl Zeiss, Tokyo, Japan). The intensity of the fluorescence in the nucleus was analyzed, as described previously [20].
Statistical Analysis
Data were analyzed by Student t-test, setting the significance at P < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Using transcriptionally active two-cell embryos at 18 h after insemination [5], we optimized the experimental conditions for the loading of embryos with BrUTP. First, the conditions for electrical permeabilization were examined. The embryos were permeabilized with two DC pulses of 1500 V/mm for various times in the presence of 10 mM BrUTP. After permeabilization, the cells were washed and incubated for 1 h in CZB medium; then the BrU incorporated into macromolecules was detected with immunofluorescence microscopy (Fig. 1 and Table 2). The fluorescence signal for BrU in the nucleus increased as the duration of the pulse increased, until reaching a plateau at 80 µsec; no increase in fluorescence intensity was observed when the duration of the pulse was increased from 80 µsec to 500 µsec. This fluorescence signal was attributable to the incorporation of BrU in the nascent mRNA, as no signal was detected when the embryos were treated with -amanitin during the electrical permeabilization and subsequent incubation (data not shown). When the concentration of BrUTP was reduced during electrical permeabilization, the efficiency of BrU incorporation decreased (data not shown). Therefore, we set two pulses of 1500 V/mm for 80-µsec duration and 10 mM BrUTP as the optimal conditions for the BrUTP loading. This treatment did not prominently affect the gene expression or the development of the embryos at later stages. Although two blastomeres were fused in some embryos, most embryos cleaved and developed to the blastocyst stage by 25 h and 72 h after electrical permeabilization, respectively (Table 3). When the expression levels of eIF-1A, cyclin-A2, MuERV, and ß-actin in the treated and untreated embryos were compared, no appreciable differences were observed for any of genes examined at 1 or 72 h after treatment (data not shown).
|
|
|
We then examined the incorporation of BrU into nascent mRNA during various incubation periods, after BrUTP loading by electrical permeabilization. After permeabilization, the embryos were washed and cultured for various periods; the incorporation of BrU into nascent mRNA was examined by immunofluorescence microscopy (Fig. 2). Strong fluorescence was observed in the nuclei of embryos incubated for 30 min after permeabilization. The fluorescence intensity remained strong at 1 h, but was decreased slightly but not significantly at 2 h; however, the fluorescence in the cytoplasm appeared to increase slightly at this time point. Thus, it appeared that the rate of BrU incorporation decreased after 2 h; for further studies, all embryos were incubated for 1 h after electrical permeabilization.
|
Recovery of mRNA after Immunoprecipitation
We synthesized rabbit -globin mRNA labeled with BrU in vitro and examined the efficiency and specificity of the immunoprecipitation of labeled mRNAs using the monoclonal antibody that recognizes BrU. After immunoprecipitation had been conducted with 50 pg of mRNA in a 30-µl reaction mixture, mRNA recovery was measured by real-time RT-PCR (Table 4). The recovery of the labeled mRNA was 37.6%, whereas it was 2.7% when unlabeled mRNA was immunoprecipitated. The nonspecific recovery of unlabeled mRNA was much reduced by the addition of a large amount of another unlabeled mRNA as a blocking agent. When 500 ng of interferon- mRNA was added to the immunoprecipitation reaction mixture, the nonspecific recovery of unlabeled -globin mRNA was reduced to 0.1%, and the specific recovery of labeled -globin mRNA was slightly increased (48.1%). Therefore, 500 ng of unlabeled interferon- mRNA was added to the immunoprecipitation mixture for all subsequent experiments. Using this protocol, a 40% recovery was also achieved for the immunoprecipitation of 5 pg of labeled mRNA, indicating that the amount of mRNA in the sample does not affect the recovery (data not shown).
|
Validation of the Procedure for the Isolation of Nascent mRNA
To verify the practical utility of our procedure for isolating nascent mRNA, we examined the changes in the abundance of eIF-1A transcripts in the BrU-labeled, immunoprecipitated mRNA fraction and in the total mRNA pool after fertilization. The amount of eIF-1A transcript in the total mRNA pool (total eIF-1A mRNA) is known to change dynamically during the late one-cell to the late two-cell stages [10]. The amount of nascent mRNA recovered by immunoprecipitation following BrUTP loading should reflect the rate of change in the abundance of the transcript in the total mRNA pool, but not the abundance itself. First, we examined the abundance of total eIF-1A mRNA. Embryos were collected every 2 h, from 14 to 26 h after insemination, and the amount of eIF-1A mRNA was estimated by semiquantitative real-time RT-PCR (Fig. 3). The results showed that the amount of total eIF-1A mRNA increased at a constant rate until 24 h, suggesting that the rate of mRNA synthesis was almost constant during this period. However, an abrupt decrease was observed at 26 h, suggesting that the rate of mRNA synthesis was reduced between 24 and 26 h.
|
We then examined the amount of eIF-1A mRNA in the immunoprecipitated fraction. Prior to the collections at 17.5, 21.5, and 25.5 h after insemination, the embryos were loaded with BrUTP and incubated for 1 h. The BrU-labeled mRNA was recovered by immunoprecipitation, and the abundance of eIF-1A mRNA was estimated by semiquantitative real-time PCR (Fig. 3). Consistent with the observed constant increase from 14 to 24 h for total eIF-1A mRNA, the amount of immunoprecipitated eIF-1A mRNA was similar at 17.5 and 21.5 h. Given that the total amount of eIF-1A mRNA was estimated to increase more than twofold between 17.5 and 21.5 h, the amount of immunoprecipitated eIF-1A mRNA appeared to reflect the rate of change in total eIF-1A mRNA rather than the amount itself. At 25.5 h, the amount of immunoprecipitated eIF-1A mRNA was markedly decreased, which was consistent with the decrease in total eIF-1A mRNA between 24 and 26 h. Comparing the amounts of total eIF-1A mRNA or immunoprecipitated eIF-1A mRNA at 17.5 and 25.5 h, the amount of total eIF-1A mRNA was estimated to be greater at 25.5 h than at 17.5 h, whereas the amount of immunoprecipitated eIF-1A mRNA was less at 25.5 h than at 17.5 h. Thus, the amount of immunoprecipitated eIF-1A mRNA reflected the rate of change in total eIF-1A mRNA but not the amount itself. These results strongly suggest that nascent mRNA was specifically recovered by immunoprecipitation following BrUTP loading.
Comparison Between the Amounts of Nascent and Total mRNA
The amounts of nascent and total cyclin-A2, MuERV, and eIF-1A mRNAs were compared by measuring the copy number of the specific cDNAs reverse transcribed from the mRNA harvested from embryos at 14–24 h after insemination. The amount of MuERV mRNA, as well as eIF-1A mRNA, is known to increase after cleavage to the two-cell stage [21, 22], whereas the amount of cyclin-A2 mRNA decreases [23]. When the total mRNAs were examined (Fig. 4a), MuERV mRNA increased until 20 h and then remained at a constant level or slightly decreased until 24 h. Cyclin-A2 mRNA decreased until 18 h and then remained at a constant level or slightly increased until 24 h. At 22.5 h, the copy numbers of the cDNAs for MuERV and cyclin-A2 indicated that the amounts of total MuERV and cyclin-A2 mRNAs were similar and that these were much higher than the amount of eIF-1A (Fig. 4b). However, the amount of nascent MuERV mRNA was lower than the amount of nascent cyclin-A2 and of eIF-1A mRNAs at 22.5 h (Fig. 4c). These results suggest that the amount of total mRNA does not always reflect the amount of nascent mRNA. Thus, the analysis of nascent mRNAs is important in the investigation of the profiles of genes transcribed during early preimplantation development.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-amanitin-treated embryos with that in the control embryos appears to be a useful way to identify the mRNAs that are newly synthesized during the treatment with the inhibitor. Indeed, a number of -amanitin-sensitive mRNAs have been identified in this way [14]. However, this procedure would identify only those mRNA species that are present in small amounts in the accumulated maternal mRNA. If the amount in the maternal pool is large, the amount of newly synthesized mRNA is relatively small and thus any differences between the -amanitin-treated and untreated control embryos would be too small to be clearly detected. The report of Hamatani et al. [14] identified relatively few mRNA species (147 mRNAs during the late one-cell and early two-cell stages, and 1733 in the late two-cell stage of 22 000 mRNA species examined), although a significant level of transcription occurs during these developmental stages [16, 24–26]. Furthermore, if an mRNA is abundant in the maternal pool, a long incubation with -amanitin would be required to detect differences between the treated and untreated embryos, which would make it impossible to analyze the changes in the expression level over a short period. It is also a serious problem that treatment with -amanitin might affect the degradation rates of maternally derived mRNAs. Therefore, to obtain profiles of newly transcribed mRNA during early preimplantation development, a method by which the nascent mRNAs can be analyzed independently of the preexisting mRNAs is needed. Thus, we endeavored to establish a method for the isolation of newly synthesized mRNA by immunoprecipitating BrU-labeled mRNA. There are two critical points that control the successful isolation of nascent mRNA. The first is the efficiency of BrUTP loading into the embryos and BrU incorporation into the nascent mRNA. We tried several methods that had been previously reported for loading cells with BrU. UTP is not efficiently incorporated into living cells unless cell membranes are permeabilized; therefore, we incubated the embryos with BrU, expecting that it would be incorporated into the embryos and metabolized to BrUTP, as is the case for BrdU [25]. However, no incorporation of BrU into macromolecules was detected by immunofluorescence microscopy. Next, we tried loading BrUTP, using detergent-mediated permeabilization [27] and lipofection [28]. Neither method yielded significant incorporation into macromolecules (unpublished), but electrical permeabilization resulted in a strong immunofluorescent signal, as shown in Figure 1. We also attempted to microinject BrUTP into the embryos [16, 29]. Although a good signal was observed by this method, the signal was not as intense as that obtained using electrical permeabilization. Furthermore, the results of microinjection were not reproducible; in some microinjected embryos, no signal was detected, whereas it was detected in all embryos after electrical permeabilization (Table 2). Consequently, we used electrical permeabilization for BrUTP loading in the present study. We confirmed that this treatment caused no detrimental effects on the development of the embryo (Table 3).
We then examined the optimal incubation time after permeabilization. Although shorter incubation times would be better for the examination of real-time synthesis of mRNA, this would also result in smaller amounts of BrU-labeled mRNA. Thus, we sought an incubation period that would allow the accumulation of detectable amounts of labeled mRNA. The immunofluorescent signal for BrU-labeled mRNA in the nucleus decreased after incubation for 2 h, as compared with 1 h, although the signal in the cytoplasm slightly increased (Fig. 2). When the amount of eIF-1A mRNA in the immunoprecipitated mRNA fraction was examined, it had clearly increased between 30 min and 1 h, but only slightly increased between 1 and 2 h (unpublished). This result was plausible because the BrUTP loaded into embryos would gradually be metabolized and degraded.
The second critical point for the successful isolation of nascent mRNA was the efficiency and specificity of the immunoprecipitation. Using BrU-labeled and unlabeled RNA synthesized in vitro, we optimized the conditions for immunoprecipitation. We could obtain nearly 50% recovery and reduce nonspecific recovery to 0.1% by adding a large amount of unlabeled, nontarget mRNA as a blocking agent (Table 4). The addition of uridine did not reduce the nonspecific recovery (unpublished), although uridine has been reported to be a useful blocking agent [30].
The percentages of uridine in the sequences would affect the number of BrU incorporated in nascent RNA. However, this would not prominently affect the recovery of mRNA. Although the number of BrU molecules incorporated per transcript may theoretically affect the efficiency of recovery, the correlation between the number of BrU incorporated and the efficiency of recovery would not be directly proportional. Theoretically, the antibody could immunoprecipitate mRNA molecules incorporating only a single BrU. In the present study, we compared the recovered amounts of the nascent mRNAs of cyclin-A2, eIF-1A, and MuERV. Although the recovered amounts were in the following order, cyclin-A2 > eIF-1A > MuERV (Fig. 4c), the numbers of uridines in these RNA molecules are 765, 867, and 1744, respectively, demonstrating that, in these cases, recovery and uridine content were not correlated.
We compared the amounts of nascent and total mRNAs by quantifying the copy number of the cDNAs reverse transcribed from these mRNA fractions and found that they were not always correlated. Although the amounts of total MuERV and cyclin-A2 were similar and much higher than that of eIF-1A, the amount of nascent MuERV mRNA was smaller than that of cyclin-A2 and of eIF-1A (Fig. 4). These data suggested that total mRNA does not always directly reflect the transcription level. When there is a large amount of accumulated mRNA, the contribution of newly synthesized mRNA is relatively minor, and only a small increase in the amount of total mRNA is observed after de novo mRNA synthesis over a short period. Thus, it is difficult to determine from measurements of total mRNA whether a gene is being transcribed or not at a given instant during early preimplantation development. Moreover, the rate of degradation should also vary with the mRNA species and time. In the mouse embryo, it is known that 
90% of maternal mRNA is degraded between the time of resumption of the first meiosis and the late two-cell stage [1] and that the rate of degradation varies widely among mRNA species [14]. If a gene is being actively transcribed, but the rate of degradation is faster than that of synthesis, the amount of total mRNA decreases; the results of a quantitation of the transcript in total mRNA could thus lead to the erroneous conclusion that the gene is not or is only rarely transcribed. In this study, the amounts of total cyclin-A2 and MuERV mRNA were similar and stayed at a constant level from 18 to 24 h after insemination (Fig. 4a). However, the amounts of the two nascent mRNAs were substantially different at 22.5 h (Fig. 4c). A possible explanation for this apparent discrepancy is that the rates of degradation of these mRNA species might differ. Cyclin-A2 mRNA might be synthesized and also degraded at a higher rate than MuERV. The amount of total cyclin-A2 mRNA rapidly decreased until 18 h and then abruptly changed, to remain at a constant level (Fig. 4c), which was indicative of its active synthesis after 18 h. It is plausible that the degradation of accumulated mRNA continued at a constant rate before and after 18 h and that active synthesis started at 18 h, although the possibility that degradation stopped at 18 h and that little synthesis occurred thereafter cannot be excluded.
In the present study, we established a method for efficiently isolating nascent mRNA. Using this method, we demonstrated that the amounts of nascent and total mRNA changed in different ways during the two-cell stage of embryonic development, indicating that analysis of the nascent mRNA is critical to obtaining profiles of the genes transcribed during the course of development. Thus, the method presented here could provide a useful tool for investigating the regulation of gene expression in early preimplantation embryos.
|
ACKNOWLEDGMENTS |
|---|
construct. |
FOOTNOTES |
|---|
Received: 11 May 2004.
First decision: 27 May 2004.
Accepted: 23 July 2004.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  M. Ohtsu, M. Kawate, M. Fukuoka, W. Gunji, F. Hanaoka, T. Utsugi, F. Onoda, and Y. Murakami Novel DNA Microarray System for Analysis of Nascent mRNAs DNA Res, August 1, 2008; 15(4): 241 - 251. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Ribet, S. Louvet-Vallee, F. Harper, N. de Parseval, M. Dewannieux, O. Heidmann, G. Pierron, B. Maro, and T. Heidmann Murine Endogenous Retrovirus MuERV-L Is the Progenitor of the "Orphan" Epsilon Viruslike Particles of the Early Mouse Embryo J. Virol., February 1, 2008; 82(3): 1622 - 1625. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-i. Kageyama, H. Liu, N. Kaneko, M. Ooga, M. Nagata, and F. Aoki Alterations in epigenetic modifications during oocyte growth in mice Reproduction, January 1, 2007; 133(1): 85 - 94. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Persson, Q. Zhang, X. Y. Wang, S. E Ravnik, S. Muhlrad, and D. J Wolgemuth Distinct roles for the mammalian A-type cyclins during oogenesis Reproduction, October 1, 2005; 130(4): 411 - 422. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
