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a Endocrinology and Reproductive Physiology Program, Department of Animal Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Depending on the species, the MET has been demonstrated to occur at a certain time after fertilization [1, 2]. It has been previously proposed that the MET in the cow starts at the transcriptional level in late 4-cell to 8- to 16-cell embryos [3, 4]. In the former study, Barnes and First [3] showed that there were 8 embryonic proteins (alpha-amanitin-sensitive) at the late4-cell stage followed by 23 new proteins at the 8-cell stage. In the latter study, high levels of transcriptional activity were detected at the 8- to 16-cell stage [4].
However, recent studies have suggested that the MET in the cow starts even earlier than previously proposed. This is based on the fact that immunoblotting experiments have shown that the hypophosphorylated form of RNA polymerase II (IIA) is present at higher levels in 2-cell-stage embryos than in 4- and 8-cell embryos, suggesting that bovine 2-cell embryos are transcriptionally competent [5]. In another study, bovine embryos were labeled with [35S]UTP, total RNA was isolated, and results showed that there was [35S]UTP incorporation into RNA, which indicated that bovine 2-cell embryos were transcriptionally active [6]. Results of these studies are consistent with those of other investigators [7, 8] who have also reported transcriptional activity in 2-cell bovine embryos. Alpha-amanitin inhibits RNA polymerase II-dependent transcription, mRNA synthesis, at the concentration used in this study [9]. [3H]Uridine has been shown to be taken up by bovine preimplantation embryos and utilized by RNA polymerase II and has been used in a number of studies, to determine transcriptional activity [8, 10, 11].
Studies in the mouse and Xenopus have shown that regulation of gene expression at the beginning of development involves a complex mechanism in which DNA replication, histone deacetylation, and enhancers play essential roles [2, 12, 13]. The first round of DNA replication appears to be critical for gene expression in the mouse by disrupting nucleosomes, thereby providing an opportunity for maternally inherited transcription factors to bind to their cognate cis-binding sequences [14, 15]. Also in the mouse, expression of some endogenous genes has been proposed to be coupled to the first round of DNA replication while others are not [12]. In this way, DNA replication changes chromatin structure in such a way as to generate a transcriptionally permissive chromatin structure. A repressive effect due to nucleosome assembly over either the enhancer, promoter, or both may require replication factor access and chromatin remodeling. Inhibition of the first round of DNA replication should abolish expression if both promoter and enhancer function requires replication. On the other hand, if only enhancer function is affected, expression would be reduced to a basal transcriptional level [14]. Another factor that affects gene expression during early development is changes in chromatin structure. It has long been shown that chromatin structure plays an essential role in transcription, in which hyperacetylation of tails of core histones disrupts histone-DNA contact and this in turn leads to a conformational change in chromatin structure that is permissive for transcriptional activity [16]. Deacetylation of histones leads to transcriptional silencing, indicating a direct link between histone acetylation and the transcriptional process [13]. Histone deacetylases are known to be inhibited by trapoxin, tricostatin A (TSA), and butyrate [17, 18]. Mouse embryos treated with butyrate showed increases in levels of reporter gene expression from reporter genes not bearing an enhancer, to a level similar to that of untreated embryos injected with an enhancer-bearing reporter gene [19]. Consistent with this, trapoxin prevented a decrease in expression of endogenous genes during the 2-cell stage [20]. Knowing how chromatin isremodeled at the onset of mammalian development is important not only for a better understanding of how the embryo progresses through the preimplantation period, but also for explaining long-lasting epigenetic effects that appear only in adults [21, 22]. Therefore, the main question is how zygotic/embryonic genes are selectively activated.
In order to understand regulation of gene expression during early embryogenesis, we must gather data from other mammalian and nonmammalian species to determine species-specific differences that occur during early development and to apply these insights into biotechnology such as cloning in the species of interest. It would be short sighted to rely on information from one species as the only model for initiation and regulation of transcription during embryo development.
In this study, we explored the effect of inhibition of the first and second rounds of DNA replication by aphidicolin on bovine zygotes and 2-cell embryos. We have determined zygotic/embryonic transcriptional and translational activity as well as whether DNA replication-dependent chromatin remodeling favors selective activation of genes necessary for continued embryo development. We also attempted to determine the effect(s) of changes in chromatin structure through histone acetylation on the pattern of gene expression during the first two cell cycles of bovine embryos.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cumulus-oocyte complexes were aspirated from small antral follicles (2- to 8-mm in diameter) from bovine ovaries obtained at a local slaughterhouse and were washed in TL (tyrode-lactate)-HEPES and matured in TCM-199, 10% fetal calf serum (FCS), sodium-pyruvate (0.2 mM), gentamycin (25 µg/ml), FSH (5 µg/ml, FSH-P; Scherring-Plough Animal Health, Kennilworth, NJ), and 1 µg/ml estradiol. Only oocytes with evenly granulated cytoplasm surrounded by multiple layers of compact cumulus cells were used in the experiments. Ten cumulus-oocyte complexes were matured per 50-µl drop of maturation medium under mineral oil at 39°C, 5% CO2 in a humidified atmosphere. Motile sperm were separated from frozen semen by density gradient centrifugation [23] and added to fertilization drops at a final concentration of 1 x 106 sperm/ml. Fertilization was performed in 50-µl drops of TALP medium containing BSA (6 mg/ml) and supplemented with penicillamine (20 µM), hypotaurine (10 µM), epinephrine (1 µM), and heparin (2 µg/ml) [24]. Twenty-four hours postinsemination (hpi), the embryos were mechanically stripped free, with a glass pipette, of cumulus cells and attached sperm, washed, and cultured in CR1-aa medium [25] under ambient conditions described above.
Inhibition Treatment Groups
In every experiment, zygotes were randomly assigned to one of the following groups: control embryos with no inhibitor, alpha-amanitin (25 µg/ml), aphidicolin (4 µg/ml), and TSA (100 nM). A control (no inhibitor added) group (n = 30) was always cultured to blastocyst stage, and the blastocyst rate was around 50%. Alpha-amanitin was used at a 25 µg/ml concentration, which was shown to inhibit more than 80% of mRNA synthesis in early bovine embryos [26]. This concentration was chosen to avoid any side effects. Aphidicolin (4 µg/ml) inhibits DNA replication in bovine [27] and mouse embryos [28]. TSA was used at two different concentrations (930 and 100 nM) that gave the same results, and the results with 100 nM were reported. Another reason for reporting the results with 100 nM TSA was because culturing embryos with 100 nM TSAcaused a slight increase in the embryo cleavage rate from 2- to 4-cell and from 4- to 8-cell embryos (unpublished results). In the same study, we also showed that TSA at 100 nM caused a reduction in the blastocyst rate. This suggested that inappropriate deacetylation/acetylation of core histones plays important roles in the control of gene expression during early embryonic development.
To determine the transcriptional and translational activity during the first and second cell cycle, zygotes (n = 150) and 2-cell embryos (n = 150) were incubated with the inhibitors from 10 hpi to 33 hpi and from 24 hpi to 44 hpi, respectively. Inhibitors were present during both protein and RNA labeling. As a control, Day 7 blastocysts (n = 3 per treatment) were cultured in the presence or absence of alpha-amanitin, aphidicolin, TSA, and cold uridine (2 mg/ml), and were labeled with [3H]uridine for 2 h. Three Day 7 blastocysts were lysed, and ribonuclease (RNase) A (100 µg/ml) was added briefly before total RNA isolation. All experiments were repeated three times.
RNA Labeling and Isolation of Total RNA
Fifty zygotes or 2-cell embryos were cultured in 50 µl of CR1-aa medium (no FCS added) supplemented with 250 µCI/ml [5,3H]uridine (specific activity 26–28 Ci/ml; Amersham, Arlington Heights, IL) for 5 h between 28 and 33 hpi, and 39 and 44 hpi for the zygotes and 2-cell embryos, respectively. After labeling, zygotes and embryos were washed four times in 3 ml TL-HEPES and stored in a -70°C freezer until processed for RNA isolation.
Total RNA was isolated using a Micro RNA Isolation kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. Briefly, zygotes and 2-cell embryos (n = 150) were frozen and thawed three times. Samples were vortexed vigorously for 3 min after adding the following into each sample tube: 100 µl of denaturing solution (4 M guanidium isothiocyanate, 0.02 M sodium citrate, and 0.5% sarcosyl), and 0.72 µl 14.4 M beta-mercaptoethanol, 10 µl 2 M sodium acetate (pH 4.0), 100 µl phenol saturated with water (pH 5.5), and 20 µl chloroform:isoamyl alcohol. The mixtures were then microfuged for 5 min (13 000 rpm), and the upper phase (containing RNA) was transferred into an RNase-free, sterile tube. One microliter of 2 mg/ml glycogen, 100 µl isopropanol, and 2 µl of 1 mg/ml carrier tRNA (Sigma Chemical Co., St. Louis, MO) were added into each sample and mixed by inversion. The samples were microfuged (13 000 rpm) at 4°C for 30 min. Supernatant was removed into another tube. The RNA pellet was washed twice with 200 µl of 75% ethanol in 0.1% diethyl pyrocarbonate (DEPC)-treated sterile water.
Determination of [3H]Uridine Incorporation and Uptake
RNA pellets were resuspended in 50 µl of 0.1% DEPC-treated sterile water, and tubes were incubated in a 60°C water bath for 5 min to facilitate RNA dissolving. Four milliliters of scintillation liquid was added, and the radioactivity was determined as counts per minute (cpm) in a Beckman LS 500TD Liquid Scintillation Counter (Beckman Instruments, Palo Alto, CA). Background incorporation was determined as cpm of unlabeled embryos or water. Uptake was determined as follows: during RNA isolation, all phases that did not contain RNA (i.e., interphases and lower phases after the first centrifugation, supernatant after the second centrifugation, and the two washing solutions of the RNA pellet) were saved into separate tubes and the cpm were determined. These counts plus the cpm of the total RNA isolated were determined. The cpm for uptake was determined by subtracting cpm of 10 µl of the last wash solution after embryo labeling (as background) from the previous cpm. The experiments were repeated three times using different pools of embryos. The data were analyzed by ANOVA,and means were compared by protected least-significance difference [29].
Protein Labeling and Two-Dimensional (2D) PAGE
Zygotes and embryos were cultured in 50-µl drops of Tyrode's albumin lactate pyruvate (TALP) medium containing BSA (6 mg/ml) and supplemented with penicillamine (20 µM), hypotaurine (10 µM), epinephrine (1 µM), and heparin (2 µg/ml) [24]. Two hours before labeling, cells were transferred into 50-µl drops of preequilibrated TALP medium under mineral oil containing the inhibitors but not penicillamine, hypotaurine, epinephrine, BSA, or FCS. To label newly synthesized zygotic and embryonic proteins, zygotes and embryos were cultured for 4 h between 29 and 33 hpi, and 40 and 44 hpi, respectively, in the FCS- and BSA-free medium supplemented with 0.1% (w:v) polyvinylpyrrolidone (Sigma Chemical Co.) and 1 mCi/ml [35S]methionine (specific activity 1000 Ci/mmol; Amersham). After labeling, zygotes and embryos were washed four times in 3 ml TL-HEPES and stored in a -70°C freezer until processed for 2D-PAGE.
Kendrick Laboratories, Inc. (Madison, WI) performed 2D-PAGE. Isoelectric focusing was carried out in glass tubes of inner diameter 2 mm, using 2.0% pH 4–8 ampholines (BDH; Hoefer Scientific Instruments, San Francisco, CA) for 9600 volts/h. After equilibration for 10 min in SDS sample buffer (190% glycerol, 50 mM dithiothreitol, 2.3% SDS, and 0.0625 M Tris, pH 6.8) the tube gels were sealed to the top of 10% acrylamide slab gels (0.75-mm thick), and SDS slab gel electrophoresis was carried out for about 4 h at 12.5 mA/gel. The following proteins (Sigma Chemical Co.) were added as molecular weight standards to a well in the agarose that sealed the tube gel to the slab gel: myosin (220 kDa), phosphorylase A (94 kDa), catalase (60 kDa), actin (43 kDa), carbonic anhydrase (29 kDa), and lysozyme (14 kDa). These standards appear as horizontal lines on the silver-stained [30] 10% acrylamide slab gels. The gels were dried between sheets of cellophane paper with the acid edge to the left. Autoradiography was carried out using Kodak X-OMAT AR film (Eastman Kodak, Rochester, NY) with an exposure of 4, 9, or 12 days at -70°C. The films were developed using Kodak developer and fixer. The experiments for 1- and 2-cell stages were conducted twice, and the same results were obtained.
Analyses of 2D-PAGE
Two experienced researchers from the Kendrick Laboratories analyzed the autoradiographs independently to determine the polypeptides that were in the control but not in the alpha-amanitin group (these groups of polypeptides would be zygotic/embryonic in origin). The presence or absence of these polypeptides was then determined for the aphidicolin and TSA groups. The autoradiographs were then scanned, pixel densities of each spot were measured, and pixel densities of the same volume of a background area were subtracted from the pixel densities of each spot by computer software (NIH, Bethesda, MD] Image Analyzer) in our lab. The pixel densities of each spot in the treatment groups were compared to the corresponding spot in the control group that had no inhibitor added. The high level of labeling was shown as "++" if the pixel densities of the treatment groups were more than 3-fold higher than those of control groups. If the differences in pixel densities of the spots in the treatment groups and the control groups were between 1- and 2-fold lower or higher, they were given "+" to indicate medium labeling; if they were 2- to 3-fold lower than that of control, they were given "±" to indicate weak labeling; and if they were more than 3-fold lower, they were given "-" to indicate no labeling. Other ways of analyzing theautoradiographs such as expressing the densities relative to densities of a reference protein (protein 12 in autoradiographs of this study) were also taken into consideration. However, the densities of this reference protein fluctuated among the experimental groups, and therefore this option was not pursued. The intensity comparisons of the spots were performed only within each cell stage, not across the two cell stages. The higher of the polypeptide spots in the 2-cell stage may be due to longer exposure of these gels to the films since these gels were exposed to the films longer than those of the 1-cell stage. Each gel was loaded with the same amount of total (soluble) cellular proteins. All of the gels for the same cell stage were exposed to films for the same periods of time, i.e., 4, 9, or 12 days, and the films that had the same exposure times were used for the analyses.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Day 7 blastocysts were used as a control since they are transcriptionally active [4]. Control blastocysts and blastocysts treated with TSA had the same level of [3H]uridine incorporation, which was significantly higher than that of the other groups (Fig. 1). Blastocysts treated with alpha-amanitin, RNase A, or cold uridine showed essentially background levels of [3H]uridine incorporation, indicating that [3H]uridine was incorporated into RNA and the assay used was reliable (Fig. 1). Zygotes cultured with TSA had the highest incorporation, while control embryos had higher incorporation than alpha-amanitin and aphidicolin groups (P < 0.05), whose incorporation did not differ from each other (P > 0.05). Patterns of [3H]uridine uptake were similar to those for incorporation in all of the groups (Fig. 2). Analyses of zygotic protein profiles are shown in Figure 3, A–D, and Table 1. Proteins numbered 1, 4, 5, 6, 7, and 8 were alpha-amanitin-sensitive, indicating that these proteins were due to translation of zygotic messages. Proteins numbered 2 and 3 were at high levels in the control zygotes, but faint in alpha-amanitin-treated zygotes, suggesting that they were from both maternal and zygotic messages. The synthesis of proteins 3, 4, and 9 was DNA replication-independent (not inhibited by aphidicolin). Proteins 5, 6, 7, and 8 were DNA replication-dependent (inhibited by aphidicolin). Synthesis of protein 11 was activated by aphidicolin since it was not expressed in the control or alpha-amanitin-treated zygotes. Aphidicolin also caused a decrease in the levels of proteins 1 and 2, and an increase in protein 10. Treatment with TSA (an inhibitor of histone deacetylases) activated expression of protein 11, caused a reduction in proteins 1–5, and inhibited expression of proteins 6, 7, and 8. Protein 12 was a reference protein whose synthesis is not affected by alpha-amanitin, aphidicolin, or TSA.
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Transcription and Translation in the 2-Cell-stage Embryos
The 2-cell control embryos without any inhibitor added had the highest [3H]uridine incorporation while the embryos cultured with TSA had higher incorporation than alpha-amanitin and aphidicolin groups (P < 0.05), whose incorporation did not differ from each other (Fig. 4). Patterns of alpha-amanitin uptake were similar to the patterns of incorporation in all of the groups.
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Embryonic proteins and their expression patterns in different treatment groups are shown in Figure 5, A–D. Proteins 5, 7, 8, and11 were alpha-amanitin-sensitive, suggesting that these proteins were due to translation of embryonic messages. Intensities of proteins 1, 2, 6, and 10 were lower in the alpha-amanitin group than in the control group, suggesting that they were from both maternal and embryonic messages. Proteins 4 and 9 had higher intensities in the alpha-amanitin group than in the control group, suggesting a higher level of translation in alpha-amanitin groups (in the absence of embryonic transcription). Proteins 5, 8, and 11 were DNA replication-dependent, i.e., inhibited by aphidicolin. Proteins 2 and 7 were DNA replication-independent, i.e., not inhibited by aphidicolin. Aphidicolin treatment caused a reduction in the levels of expression of proteins 1 and 6, and an increase in proteins 3, 4, and 10. Inhibition of histone deacetylases by TSA inhibited expression of proteins 2, 5, 6, 8, 10, and 11; reduced expression of protein 1; and increased expression of proteins 3, 4, and 9.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It has been proposed that the MET occurs at the 8- to 16-cell stages in bovine embryos [4]. We have previously shown that the expressed MET begins with 8 protein products of the embryonic genome at the late 4-cell stage and that bovine embryonic transcription begins as early as the 2-cell stage [3, 6]. Other studies have also suggested that bovine 2-cell embryos are transcriptionally active [8, 11]. Therefore, we asked whether there is any transcriptional activity in 1-cell-stage zygotes. Our objective here was to better understand how this early embryonic transcription during the first and second cell cycles was initiated in terms of timing and mechanism. Our previous autoradiographic studies indicated that this early transcriptional activity at the 2-cell stage was from nuclear transcription [6]. We analyzed transcriptional activity during the first cell cycle, culturing 1-cell zygotes in the presence or absence of alpha-amanitin, aphidicolin, and TSA, and labeled with [3H]uridine for 5 h between 29 hpi and 34 hpi. Transcriptional activity in all of the groups was significantly higher than background (P < 0.05; Fig. 2). The origin of [3H]uridine incorporation in alpha-amanitin groups may be incorporation into tRNA since the synthesis of tRNA is believed to be turned on as embryonic genes are activated (Dr. Richard M. Schultz, personal communication), and alpha-amanitin at this concentration does not inhibit tRNA synthesis. Another origin of [3H]uridine incorporation in alpha-amanitin groups may be into mRNA that is not inhibited by alpha-amanitin at this concentration (
15%, see inhibition treatment groups). Ribosomal RNA synthesis has been shown to start at the 8-cell stage in the cow [4]. Therefore, [3H]uridine incorporation during 1- and 2-cell stages should not be into rRNA.
The inhibitors used—aphidicolin, alpha-amanitin, and TSA—have been shown to specifically inhibit DNA replication, mRNA synthesis, and histone deacetylases, respectively (see references in Introduction). There have been no reports indicating that the inhibitors affect uridine uptake. Uridine uptake by bovine embryos has been reported in a number of studies [8, 10, 11]. Therefore, it is unlikely that the inhibitors affect only uptake. The lowest uridine uptake in this study was 10 000, and the incorporation was 100. Even in the cells that had the lowest uptake, there was still an abundant amount of uridine taken up by the cell that could be incorporated. Therefore, uptake atthe levels of this study should not affect incorporation; rather the amount of incorporation is a result of inhibition of mRNA, DNA replication, or histone deacetylases (also see the control experiments with blastocysts treated with or without cold uridine or RNase A). Therefore, incorporation determines the level of uptake.
Analysis of the protein profiles in each treatment showed that there are 6 proteins present in the control zygotes that are not present in the alpha-amanitin-treated zygotes (Fig. 3, A and B, and Table 1; proteins 1, 4–8), suggesting that these are due to translation of zygotic messages. There were also two proteins (proteins 2 and 3) whose levels were higher in the control group than in the alpha-amanitin group, suggesting that these proteins are due to translation of both maternal and zygotic mRNAs. Since 2-cell embryos are transcriptionally active [6, 11, 31], we therefore aimed to analyze this early gene expression at both the transcriptional and translational levels using the same approaches as in the zygotes. Analyses of the protein profiles for the 2-cell-stage embryos showed that the majority of the polypeptide spots remained the same between 1- and 2-cell stages. There were fewer alpha-amanitin-sensitive proteins in the 2-cell embryos than in the 1-cell zygotes (Fig. 5, A and B, and Table 1). This may be because of translation of maternal messages of those proteins in the absence of embryonic transcription. Some of the proteins in the alpha-amanitin group were present at lower levels, suggesting that these proteins are both maternal and zygotic/embryonic in origin. Results of this study and previous results in our laboratory [6, 32], and data obtained by others [11, 33] suggest that the MET in the cow is characterized by a minor gene activation between the 1- and 4-cell stages, and a major gene activation at the 8- to 16-cell stages [3, 4]. Our previous studies also showed that this early yet minor gene activation is essential for embryo development as zygotes and 2-cell embryos exposed to alpha-amanitin during the first and second cell cycles cannot develop beyond the 9- to 16-cell stage [5].
Effect of Inhibiting DNA Replication on Transcriptional and Translational Activity in 1-Cell Zygotes and 2-Cell Embryos
DNA replication has been proposed to provide access for maternally derived transcriptional factors to bind their DNA binding sites [12], and disrupt transcriptionally repressive nucleosomes [15], both of which result in activation of transcription. To explore the effect of inhibition of the first and second rounds of DNA replication on bovine zygotic/embryonic transcription and translation, zygotes and 2-cell embryos were cultured with aphidicolin, an inhibitor of DNA polymerases [34] until 33 and 44 hpi, the time that corresponds to the 2- and 4-cell stages, respectively, and were labeled with [3H]uridine (between 29 and 34 hpi, and 40 and 45 hpi, respectively) or with [35S]methionine between 29 and 33 hpi, and 40 and 44 hpi, respectively. Inhibition of the first and second rounds of DNA replication decreased transcriptional activity both in zygotes and in 2-cell embryos (Figs. 2 and 4). These results suggest that DNA replication may be required for an optimal level of transcription during the first and second cell cycles. We detected a group of several zygotic/embryonic proteins whose synthesis during the first and second cell cycles appeared to be dependent on the first and second rounds of DNA replication (replication-dependent proteins whosesynthesis is coupled to the first and second rounds of DNA replication). We also detected another group of zygotic/embryonic proteins whose synthesis during the first and second cell cycles did not depend on the first and second rounds of DNA replication (Fig. 3, A–C, and Table 1). Similar results have been reported in 2-cell mouse embryo [14]. Data presented here suggest that the first and second rounds of DNA replication are important for endogenous transcriptional activity and control of gene expression at the onset of bovine embryonic development. Decreased transcriptional activity in aphidicolin-treated zygotes and 2-cell embryos in this study also suggests that the first and second rounds of DNA replication do not suppress transcription. A requirement for the first round of DNA replication for maximum expression of translation initiation factor eIF-4C and transcription-requiring complex has been demonstrated in the mouse embryo [14]. DNA replication-dependent remodeling of chromatin may selectively activate genes necessary for further embryo development [20]. Proteins 10 and 11 at the 1-cell stage and 9 and 10 at the 2-cell stage had increased levels of labeling when DNA replication was inhibited with aphidicolin. This is consistent with the results obtained by other studies of bovine embryos [27], in which 8-cell specific cdc25 mRNA expression was induced in 2-cell embryos upon treatment with aphidicolin. As mentioned previously, expression of some of the proteins was inhibited totally, some were partially inhibited, and yet others were not affected at all or were even activated (Fig. 3, A–C, and Table 1). This may be explained by repressive effects of nucleosome assembly over either enhancer, promoter, or both [20]. In summary, DNA replication during the first and second cell cycles may be required for zygotic/embryonic transcription and regulation of gene expression.
Effect of Inhibiting Histone Deacetylases on Transcriptional and Translational Activity in 1-Cell Zygotes and 2-Cell Embryos
Developmental control of gene expression is influenced by chromatin structure and biochemical composition of individual nucleosomes [35]. Posttranslational changes in histones such as acetylation and phosphorylation affect transcription of mRNA. Turner and Fellows [36] showed that in mammalian somatic cells, histone H4 is acetylated in the order lysine 16, followed by lysine 8 or 12, and then by lysine 5. When core histones are acetylated, chromatin loosens and thereby provides an opportunity for transcription initiation factors to be able to bind DNA. Similarly, some integral components of the transcription initiation complex such as the SWI/SNF protein complex (subunit of RNA polymerase II holoenzyme) have been shown to change the chromatin structure when they bind to transcription factors such that this also provides the transcription initiation complex the opportunity to start transcription and displace nucleosomes during transcriptional elongation [37, 38]. Transcriptionally repressive chromatin structure can be due to the presence of a complete histone octamer, low acetylation of core histones, and/or the presence of a linker histone such as histone H1 [28]. Repression of transcription in mouse embryos coincides with the appearance of histone H1 and a decrease in histone H4 hyperacetylation [39]. In this study, zygotes treated with TSA (inhibitor of histone deacetylases) had the highest transcriptional activity compared to that in the other groups (Fig. 2). These high levels of transcriptional activity suggest that relieving repressive chromatin structure may permit activation of transcription in zygotes. What the levels of transcriptional activities are, and what mechanism(s) regulate the transcriptional activity in 1-cell-stage zygotes (in both male and female pronuclei), are intriguing questions yet to be answered in the cow. Studies in mice have shown that the male pronucleus has higher transcriptional activity than the female pronucleus because of hyperacetylation of histones in the male pronucleus [12, 21]. In our study, inhibition of histone deacetylases in the 2-cell bovine embryos treated with TSA did not cause an increase in transcriptional activity (Fig. 4) compared to that in the control embryos. This may be due to the fact that the genes activated at this cell stage may not be responsive to the changes in acetylation of histones. Alternatively, it is possible that the chromatin in this cell stage may not be transcriptionally repressive. Zygotes and 2-cell embryos treated with TSA had significantly higher transcriptional activity than alpha-amanitin and aphidicolin groups (P > 0.05; Figs. 2 and 4). Even though a few proteins had an increased level of labeling, expression levels of some zygotic/embryonic proteins decreased when histone deacetylases were inhibited with TSA (Fig. 3D and Table 1). The reduction in protein expression may be due to an inappropriate decrease in the levels of histone deacetylases or an increase in histone acetyltransferases since the levels of these two enzymes are in equilibrium in a normal situation [40]. Yoshida et al. [17] showed that inhibition of histone deacetylases by TSA induces histone hyperacetylation. However, Dimitrow et al. [41] have suggested that in Xenopus, histone acetyltransferase activity is developmentally regulated since accumulation of histone acetyltransferases occurred in the presence of TSA only during early gastrulation. Moreover, Xenopus embryos cultured in TSA had defective mesoderm [35], and in the starfish Asterina pectinifera, embryo development was arrested in early gastrula stage when histone deacetylases were inhibited [42]. Therefore, it is possible that only a small fraction of the total genome is affected by TSA. Developmental control of histone acetylation and deacetylation during early bovine embryogenesis is currently under investigation. In summary, many lines of evidence suggest that changes in chromatin structure due to acetylation of core histones play an important role in zygotic/embryonic gene activation and regulation of gene expression for continued embryo development [43, 44].
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Neal L. First, University of Wisconsin-Madison, Department of Animal Sciences, 1675 Observatory Drive, Madison, WI 53706. FAX: 608 262 5157; nlf{at}calshp.cals.wisc.edu
3 Current address: Harvard Medical School, Harvard Institute of Human Genetics, 4 Blackfan Circle, Boston, MA 02115.
Accepted: June 10, 1999.
Received: February 22, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 M. Vallee, C. Gravel, M.-F. Palin, H. Reghenas, P. Stothard, D. S. Wishart, and M.-A. Sirard Identification of Novel and Known Oocyte-Specific Genes Using Complementary DNA Subtraction and Microarray Analysis in Three Different Species Biol Reprod, July 1, 2005; 73(1): 63 - 71. [Abstract] [Full Text] [PDF]
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A. Oropeza, C. Wrenzycki, D. Herrmann, K.-G. Hadeler, and H. Niemann Improvement of the Developmental Capacity of Oocytes from Prepubertal Cattleby Intraovarian Insulin-Like Growth Factor-I Application Biol Reprod, June 1, 2004; 70(6): 1634 - 1643. [Abstract] [Full Text] [PDF]
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C. Vigneault, S. McGraw, L. Massicotte, and M.-A. Sirard Transcription Factor Expression Patterns in Bovine In Vitro-Derived Embryos Priorto Maternal-Zygotic Transition Biol Reprod, June 1, 2004; 70(6): 1701 - 1709. [Abstract] [Full Text] [PDF]
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S. Gao, E. Czirr, Y. G. Chung, Z. Han, and K. E. Latham Genetic Variation in Oocyte Phenotype Revealed Through Parthenogenesis and Cloning: Correlation with Differences in Pronuclear Epigenetic Modification Biol Reprod, April 1, 2004; 70(4): 1162 - 1170. [Abstract] [Full Text] [PDF]
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A.S. Lequarre, J. Marchandise, B. Moreau, A. Massip, and I. Donnay Cell Cycle Duration at the Time of Maternal Zygotic Transition for In Vitro Produced Bovine Embryos: Effect of Oxygen Tension and Transcription Inhibition Biol Reprod, November 1, 2003; 69(5): 1707 - 1713. [Abstract] [Full Text] [PDF]
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P. Comizzoli, F. Urner, D. Sakkas, and J.P. Renard Up-Regulation of Glucose Metabolism During Male Pronucleus Formation Determines the Early Onset of the S Phase in Bovine Zygotes Biol Reprod, May 1, 2003; 68(5): 1934 - 1940. [Abstract] [Full Text] [PDF]
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 J. Ramalho-Santos, P. Sutovsky, C. Simerly, R. Oko, G. M. Wessel, L. Hewitson, and G. Schatten ICSI choreography: fate of sperm structures after monospermic rhesus ICSI and first cell cycle implications Hum. Reprod., December 1, 2000; 15(12): 2610 - 2620. [Abstract] [Full Text] [PDF]
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-amanitin, aphidicolin, and TSA on the pattern of protein synthesis in bovine zygotes and 2-cell embryos.*
