Biol Reprod
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, Q.
Right arrow Articles by Latham, K. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, Q.
Right arrow Articles by Latham, K. E.
Agricola
Right arrow Articles by Wang, Q.
Right arrow Articles by Latham, K. E.
Biology of Reproduction 62, 969-978 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

Translation of Maternal Messenger Ribonucleic Acids Encoding Transcription Factors During Genome Activation in Early Mouse Embryos1

Qingxue Wanga, and Keith E. Latham2,,a,b

a The Fels Institute for Cancer Research and Molecular Biology and b Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Embryonic genome activation (EGA) in mice is sensitive to treatment with cycloheximide, indicating that protein synthesis plays an important role in mediating EGA. We hypothesized that regulated maternal mRNA recruitment may control the time of EGA by controlling the time of appearance of certain transcription factors (TFs). We also hypothesized that synthesis of other TFs may contribute to EGA independently of controlling the timing of EGA. To test these hypotheses, we used sucrose density gradient fractionation coupled to a quantitative reverse transcription-polymerase chain reaction method to compare polysomal mRNA abundances of specific TF mRNAs between metaphase II oocytes, 1-cell-stage embryos, and 2-cell-stage embryos. We observed a 2-cell-stage-specific increase in polysomal abundance of mouse TEA DNA binding domain 2 (mTEAD-2) mRNA, coincident with the first appearance of mTEAD activity in the early embryo. The mRNAs encoding Sp1, TATA binding protein, and cyclic AMP response element binding protein did not undergo translational recruitment, but exhibited differences in polysomal abundance. We also observed a continuous, high proportion in the polysomal fraction for the mRNA encoding ribosomal protein L23 mRNA, which contrasted with the patterns observed for other maternal transcripts. These observations are consistent with the hypothesis that regulated recruitment of maternal TF mRNAs may control the time of activation of some genes during EGA, and that continuous synthesis of other TFs, like Sp1, may facilitate EGA.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In metazoan animals, fertilization produces a 1-cell embryo that contains a haploid set of maternal chromosomes and a haploid set of paternal chromosomes united within a common cytoplasm. Both parental genomes are contributed via two highly differentiated cells, the sperm and egg. After fertilization, the primary function of the oocyte is to transform the maternally and paternally inherited genomes into a single, totipotent embryonic genome that possesses the necessary epigenetic as well as genetic information to execute the developmental program correctly. In mammals, epigenetic modifications of the maternal and paternal genomes during the immediate post-fertilization period arise through a complex set of processes that collectively form the pronuclei or embryonic nuclei, remodel chromatin structure, and impose specific modifications on the paternal genome [1, 2].

Data from nuclear transplantation studies indicate that transcription of the zygotic or embryonic genomes must be delayed until the nuclear remodeling and reprogramming events that follow fertilization have been completed [3]. For example, transcriptionally active mouse 8-cell-stage nuclei exposed to 1-cell cytoplasm undergo gene repression, and some genes remain incorrectly repressed in 2-cell-stage nuclear transplant embryos [4]. Therefore, at least some genes within transcriptionally active genomes can be inappropriately modified and silenced when exposed to early 1-cell cytoplasm. The early period of transcriptional silence may serve a protective function during normal embryogenesis by limiting the access of modifying factors to genetic material, thereby avoiding inappropriate epigenetic modifications. Consequently, mechanisms should exist that delay transcriptional activation until essential nuclear reprogramming events have been completed.

The need to delay transcriptional activation of the embryonic genome (EGA) until essential nuclear events have occurred must be balanced with metabolic requirements of the developing embryo. The maternal endowment of proteins and mRNAs in mouse embryos decays during the 1-cell and 2-cell stages. As a result, essential macromolecules that support metabolic needs of the embryo may become limiting. In addition, de novo gene expression is likely to be required in order to initiate the correct developmental program.

In the mouse embryo, these apparently conflicting needs appear to be balanced by the development of a "transcriptionally permissive" state during the late 1-cell stage. Several lines of evidence have revealed the development of a transcriptionally permissive state in the 1-cell mouse embryo. One line of evidence was the ability of late, but not early, 1-cell cytoplasm to support transcription in exogenous nuclei. The early 1-cell-stage embryonic cytoplasm does not support transcription when {alpha}-amanitin-inhibited, but otherwise transcriptionally competent, 2-cell-stage nuclei are introduced by nuclear transplantation [5]. Thus, the early 1-cell cytoplasm either contains one or more factors that can enter the donor nuclei and inhibit transcription, or lacks a sufficient amount of active RNA polymerase II to restore transcription to the {alpha}-amanitin-inhibited donor nuclei. Late 1-cell-stage cytoplasm, however, permits gene transcription in such nuclei, indicating that it is able to contribute the required RNA polymerase II and is not generally transcriptionally repressive [5]. Other studies revealed the ability of 1-cell-stage embryos to transcribe exogenous reporter genes, transgenes, and endogenous genes [6–10]. Although some genes can be transcribed during the late 1-cell stage, the major genome activation event in mice is delayed until the 2-cell stage [11–17]. Thus, after an initial transcriptionally silent period, during which extensive remodeling and reprogramming can occur, some genes are permitted to be transcribed in the late 1-cell embryo, but most genes remain silent until the 2-cell stage. In other species of mammals, the same situation is likely to exist, but with differences in the actual timing of nuclear and transcriptional events [18, 19]. Thus, the mammalian embryo presents the interesting regulatory challenge of severely limiting global transcriptional activity, even during a period in which the cytoplasm is transcriptionally permissive and a subset of genes becomes active.

We hypothesize that a major controlling mechanism that limits transcriptional activity in the early embryo is the absence or paucity of essential transcription factors (TFs). This hypothesis is based on the finding that inhibiting protein synthesis from the late 1-cell stage onward inhibits transcription-dependent expression at the 2-cell stage of all housekeeping genes tested, to about the same degree as {alpha}-amanitin treatment [20]. We further hypothesize that some essential TFs may appear in a developmentally regulated manner, because of translational recruitment of the corresponding maternal (and possibly zygotic) mRNAs, and that other essential TFs may be somewhat unstable and require continuous synthesis in order for widespread EGA at the 2-cell stage to occur. To test these hypotheses, we have used a quantitative reverse transcription (RT)-polymerase chain reaction (PCR) method coupled to sucrose density gradient fractionation to determine the temporal patterns with which mRNAs encoding different TFs in the mouse embryo are associated with polyribosomes, as an indicator of active mRNA translation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Embryo Culture

Unfertilized metaphase II oocytes (MII oocytes) and fertilized 1-cell embryos were isolated from superovulated Swiss female mice (National Institutes of Health [NIH], Bethesda, MD) mated to either (B6xSJL)F1 or (B6D2)F1 male mice (The Jackson Laboratory, Bar Harbor, ME, or Taconic, Germantown, NY, respectively). Mice were superovulated by administration of 5 IU eCG followed 46–48 h later by 5 IU hCG. Unfertilized MII oocytes were lysed immediately in polysome lysis buffer (PLB, see below). One-cell embryos were cultured in 24 µg/ml {alpha}-amanitin in potassium simplex optimized medium (KSOM) + amino acids [21] at 37°C in a humidified atmosphere containing 5% CO2, 5% O2, and 90% N2. One-cell embryos were then lysed in PLB at 29 h post-hCG. Two-cell embryos were obtained by further culturing of 1-cell embryos until approximately 45 h post-hCG. The use of {alpha}-amanitin ensured that any changes in distribution between polysomal and nonpolysomal fractions observed reflected changes in the distribution of maternally derived transcripts within the embryo, as opposed to shifts that might result from the early expression of zygotic transcripts. In some experiments, 2-cell-stage embryos not treated with {alpha}-amanitin were also analyzed.

Polysome Gradient Analysis and mRNA Isolation

For sucrose density gradient fractionation, MII oocytes and embryos (~500–1000) were lysed in 300 µl PLB (20 mM Tris, pH 7.5, 5 mM magnesium acetate, 100 mM NH4Cl, 5 mM dithiothreitol, 1% Nonidet P-40, 0.5% deoxycholate, 2000 U/ml RNA-Guard [Pharmacia, Piscataway, NJ] and 100 U/ml Prime RNase Inhibitor [5 Prime->3 Prime; Boulder, CO]) and were homogenized by several passages through a 27-gauge needle. The lysates were centrifuged at 12 000 x g at 4°C for 15 min to remove nuclei and mitochondria. The post-mitochondrial supernatants were layered on 5-ml sucrose gradients (15–40% w:v), and centrifuged at 36 000 x g for 2 h. Gradient fractions (0.5 ml) were collected. The bottom four fractions were taken as polysomal, the next two intermediate fractions as monosomal-trisomal, and the remaining upper fractions as nonpolysomal. Within each of the three gradient zones (polysomal, intermediate, and nonpolysomal) the fractions were mixed together, adjusted to 25 mM EDTA, and extracted two times with phenol:chloroform:isoamyl alcohol (49.5:49.5:1; Fluka, Ronkonkoma, NY) and once with chloroform. Thus, for each gradient, between 2 and 4 samples were obtained for each gradient zone. Between two and four independent gradients were obtained for each stage analyzed. After phenol extraction, each gradient fraction was diluted 1:1 with ribonuclease (RNase)-free water. The mRNA was precipitated overnight at -20°C by addition of polyC (Boehringer-Mannheim Biochemicals, Indianapolis, IN) as carrier (40–50 µg/ml), sodium acetate to 0.3 M, and isopropanol to 40%. RNA pellets were recovered, extensively washed with 75% ethanol, and dissolved in RNase free water containing Prime RNase Inhibitor (1:100 dilution) and used for RT.

Quantitative RT-PCR Analysis

Quantitative RT-PCR analysis was performed using the quantitative amplification and dot blotting (QADB) method as described [22]. With this method, the 3' termini of the entire mRNA population are amplified, and the relative abundances of different mRNAs are preserved in the sequence abundances of the cDNA product. Expression patterns for individual mRNAs are then determined by quantitative hybridization analysis using dot blots produced with the amplified cDNAs.

For characterization of temporal expression patterns, blots containing samples corresponding to germinal vesicle (GV)-stage oocytes, MII oocytes, and embryos at the 1-cell through blastocyst stages were used as described [22]. For analysis of mRNA translation patterns, the RNA pellets obtained from the sucrose density gradient separations were dissolved in RNase-free water supplemented with Prime RNase Inhibitor as described above. The RNA was then subjected to RT-PCR as described [22] except that RT was performed in the absence of added NP-40. The amplified cDNA was then analyzed by dot blotting and hybridization as described [22]. Hybridization signals were quantified by phosphorimaging and normalized to the amount of amplified cDNA bound to each dot as described [22]. The average hybridization signal for each gradient zone was calculated and expressed as a percentage of the sum of the averages for the three zones. For each of the three gradient zones, the percentage values for the independent gradients were then averaged together.

Hybridization probes used in the study included those for four transcription factors (Sp1, TATA binding protein [TBP], mouse TEA DNA binding domain 2 [mTEAD-2], and cyclic AMP response element binding protein [CBP]), and two other mRNAs expressed as maternal transcripts (mouse elongation factor 1-alpha [EF1{alpha}] and ribosomal protein L23 [rpL23]). The cDNA probe for Sp1 was a 445-base pair (bp) terminal AflIII-HindIII fragment isolated from the pMusSp1–11 plasmid [23]. A cDNA probe for TBP was obtained by RT-PCR, and its identity was confirmed by DNA sequencing (PCR primers: 5'-CTCAGTTACAGGTGGCAGC-3' and 5'-CTGGGAAGGCGG AATGTATC-3'). The probe for the CBP mRNA was the generous gift of Dr. Elizabeth Moran and Dr. Peter Dallas (The Fels Institute, Philadelphia, PA). The cDNA for mTEAD-2 was the generous gift of Dr. Mel DePamphilis and Dr. Kotaro Kaneko (NIH, Bethesda, MD), and consisted of a 650-bp XhoI/DraI fragment as described [24]. The cDNA for EF1{alpha} was the generous gift of Dr. Susannah Varmuza (University of Toronto, Toronto, ON, Canada) [25]. The cDNA for rpL23, a 290-bp partial cDNA clone corresponding to the 3' terminus, was isolated from a mouse blastocyst stage library, and its identity was determined by DNA sequencing. The cDNA for beta globin used in assay validation studies was produced by RT-PCR from purified globin mRNA (Gibco-BRL, Rockville, MD) using primers as described [26].

Validation of the QADB RT-PCR Assay

Several different methods of quantitative and semi-quantitative RT-PCR assays have been developed and applied to mammalian embryos, each with distinct advantages and limitations. Other than the QADB method employed here, the two major methods employed have been the single nucleotide primer extension (SNuPE) method described by Singer-Sam and coworkers (e.g., [27]) and the semi-quantitative RT-PCR method developed by Richard Schultz and coworkers [26]. Both of these alternative methods of analysis have proven very useful, but are subject to experimental limitations that the QADB method was designed to circumvent. The SNuPE method is very sensitive and can be applied to small amounts of material, but is laborious when a standard curve is used for quantitation, and typically yields data for only one or a few mRNAs from a single sample. The semi-quantitative method requires larger amounts of material to achieve efficient RT and amplification, produces data for a small number of mRNAs, and does not provide information about actual mRNA abundance. The QADB method, by contrast, is applicable to small amounts of material, even single embryos or single cells, provides the ability to quantify expression of a large number of mRNAs, and in some situations can provide estimates of actual mRNA abundance. These properties make the QADB method ideal for examining mRNA abundances in sucrose density gradient fractions of oocyte and embryo lysates.

The QADB method has been used extensively by our laboratory [20, 22, 24, 28–37]. In addition, the RT-PCR method used to generate the cDNA before quantitative blot hybridization analysis has been used extensively in the preparation of representative cDNA libraries and in other quantitative studies based on single-cell analyses (e.g., [38–41]). Although differences in experimental design and assay sensitivity limit the degree to which embryological studies from different laboratories may be compared, comparisons of published data indicate that the QADB has generally yielded expression data that are in good agreement with data obtained by alternative methods. For example, temporal patterns of expression observed for a number of transcripts (e.g., tPA, Hprt, G protein {alpha}S, {alpha}q, {alpha}i2, {alpha}13, {alpha}11, and {alpha}14 isoforms, c-myc, Pgk1, U2afbp-rs, Bcl-2, Bax, Bcl-x, Bad, Bcl-w, Caspase-2, Na+/K+-ATPase {alpha}1 subunit) are very similar (after accounting for differences in frequency of sampling, the number of time intervals assayed, and the quantitative resolution of assay methods used) to the patterns observed with other methods, such as RT-PCR, Northern blotting, and Western blotting, or in some cases to the inferred pattern of embryonic gene expression based on the results of experimental treatments such as the use of antisense oligonucleotides (compare above references with the following: tPA, [42–44] Hprt, [45]; G proteins, [46]; c-myc, [47, 48]; Pgk1, [27]; U2afbp-rs, [49]; Bcl-2, Bax, Bcl-x, Bad, Bcl-w, Caspase-2, [50, 51]; Na+/K+-ATPase {alpha}1 subunit, [52, 53]). The QADB method has even been able to detect the low-abundance Xist RNA expression at the 2-cell stage in mouse embryos [31], consistent with XIST RNA expression in the early (4-cell-stage) human embryo [54] and Xist promoter-driven expression of a transgene in mouse 2-cell embryos [55]. Another study employing a different RT-PCR method was unable to detect Xist expression in 2-cell embryos [56]. The patterns of expression observed for different mRNAs during preimplantation development generally exhibit smooth profiles and comparatively small standard errors, indicating good reproducibility in the data. The magnitude of changes in estimated specific mRNA abundance observed with the QADB method has been similar to that derived by other methods of analysis [22]. In addition, the use of the QADB method to examine expression of imprinted genes has revealed the expected effects of parental chromosome origin on the preimplantation expression of the imprinted Mash-2, U2afbp-rs, H19, Xist, and Snrpn genes, including approximately 2-fold greater levels of expression in the appropriate uniparental embryos as compared with control fertilized embryos. The quantitative estimates of mRNA copy number produced for embryos by the QADB method also are in good agreement with other estimates obtained, for example, by Northern blotting [22, 34].

To test the quantitative power of the QADB method, we performed an analysis of a dilution series of rabbit beta globin mRNA added to total RNA from mouse HC11 cells. The results (Fig. 1) reveal that the QADB method produced hybridization signals that were linear over at least three orders of magnitude (R2 = 0.992) and extended to a very low abundance of the globin mRNA. Although the actual lower limit of detection of the assay has not been exhaustively tested, these data combined with data from analyses of rare transcripts (e.g., Xist [31]) in mouse embryos indicate that the method is highly sensitive and quantitative.



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 1. Quantitative aspects of the QADB method. A dilution series of rabbit globin mRNA was prepared as a mixture with total RNA from mouse HC11 cells. The QADB method was applied using a rabbit beta globin cDNA probe. Hybridization signals were normalized to the endogenous EF1{alpha} mRNA. Points reflect the mean measurement (± SD). Globin mRNA concentrations are given as picograms of globin mRNA per micrograms of total HC11 cell RNA

As a test of the quantitative reproducibility of the QADB method, we evaluated the reproducibility of hybridization signals obtained for a series of samples representing single-embryo equivalents of RNA from a combined pool of RNA from 20 blastocysts. QADB analysis for the actin mRNA indicated excellent reproducibility, with a mean value (PhosphorImager units) of 12 883 ± 506 (SEM) for 16 single-embryo equivalents.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Separation of Polysomal mRNAs

Preliminary studies with somatic cell lysates, employing continuous U.V. absorbance monitoring and use of EDTA to dissociate polysomes (data not shown), confirmed that the bottom 4 fractions contained polysomal material. In addition, sucrose density gradient fractionation was applied to lysates (intact and EDTA-treated) from small numbers of fibroblasts (~9000) to mimic the small amount of material typically obtained with oocytes or embryos. The gradient fractions were processed individually as described above (but without pooling gradient fractions) and then subjected to RT-PCR. The amplified cDNA was then subjected to agarose gel electrophoresis to visualize the products, and then transferred to a hybridization membrane by capillary blotting. Hybridization of the resulting blot to a cDNA probe for actin revealed that EDTA treatment, which is expected to dissociate the polysomes, released material from the bottom fractions (Fig. 2A). In another preliminary experiment, a lysate from GV-stage intact oocytes was analyzed in this manner (Fig. 2B). Strong hybridization signals were obtained with the bottom four fractions using an actin cDNA probe. A cDNA probe for tissue plasminogen activator (tPA) hybridized most strongly to the upper fractions. Because the tPA mRNA exists predominantly as a nontranslated maternal mRNA in GV-stage mouse oocytes [57], we designated the bottom-most four fractions (which hybridized strongly with actin probe) as polysomal, in order to minimize the chance of contamination with sub-polysomal material.



View larger version (72K):
[in this window]
[in a new window]
  		FIG. 2. Separation of polysomal mRNAs by sucrose density gradient centrifugation. A) Fractions were collected from sucrose density gradients that received lysates of ~9000 fibroblasts, either untreated or supplemented with EDTA to 0.1 M before loading. The RNA from each fraction was then purified and subjected to RT-PCR. Equal volumes of the PCR reactions were resolved by agarose gel electrophoresis, blotted, and hybridized with a cDNA probe encoding actin. Fractions labeled B, M, and T, correspond to polysomal (bottom), monosomal/trisomal (middle), and nonpolysomal (top), respectively. Note that EDTA treatment released the mRNAs from the bottom polysomal fractions. B) A lysate of ~500 GV-stage mouse oocytes was analyzed as above, and the blot was hybridized to detect the tPA mRNA and actin mRNAs. Only the bottom-most 8 fractions were processed for analysis in this experiment. Note that the tPA mRNA is predominantly localized to the sub-polysomal fractions as expected

Temporal Expression Patterns of TF mRNAs

The expression patterns for the mRNAs analyzed in this study are shown in Figure 3. All of these mRNAs are expressed in the egg as maternal mRNAs. The expression pattern of the mTEAD-2 mRNA has been reported elsewhere [24] and is reproduced here.



View larger version (41K):
[in this window]
[in a new window]
  		FIG. 3. Expression patterns of TF and other maternal mRNAs during preimplantation development. Dot blots containing a series of cDNA samples from GV-stage oocytes, MII oocytes, and preimplantation-stages embryos were hybridized to the indicated cDNA probes. Hybridization signals were normalized to the amount of DNA bound to each dot. Data are expressed in units of counts per minute bound (at left) or estimated number of copies per embryo or oocyte (at right). Copy number estimates were calculated as described [22] by accounting for cDNA probe specific activity, poly(A) mRNA content for each stage, and the estimated abundance of the actin mRNA as an internal standard. Bars indicate the mean value (± SEM) obtained for the stages or times indicated (between 3 and 8 samples per stage). Morphological stages (GV-stage oocyte, egg, 1-cell through 8-cell stages, morula, and blastocyst) are indicated above the graphs

In addition to samples of developing embryos that reveal temporal expression patterns for mRNAs analyzed, the dot blots employed in these studies contained samples of {alpha}-amanitin-treated embryos for several time points. These samples of {alpha}-amanitin-treated embryos provide a useful means for obtaining an estimate of the relative proportion of a given mRNA at a given stage that is attributable to embryonic gene transcription versus maternal mRNA deposited in the oocyte during oogenesis. Because our studies seek to examine the temporal changes in polysomal content for different maternal mRNAs, such knowledge is useful for determining which mRNAs are expressed as maternal messages and when embryonic transcription may begin to contribute to the expression observed. The abundance of mRNAs expressed predominantly as maternal transcripts should not be substantially reduced by {alpha}-amanitin treatment.

The mTEAD-2 mRNA was exclusively maternally derived throughout the period analyzed (Table 1). The Sp1 mRNA was maternally derived at 29 h post-hCG. The slight apparent increase in Sp1 mRNA abundance in treated embryos most likely reflects a greater relative stability of the Sp1 mRNA relative to the bulk of the maternal mRNA population, which undergoes decay during the 1-cell and 2-cell stages. Treatment with {alpha}-amanitin produced a more than 4-fold decrease in Sp1 mRNA abundance at 39 h post-hCG (mid 2-cell stage) and a more than 6-fold decrease at 49 h (late 2-cell stage), indicating that the majority of Sp1 mRNA normally expressed at these two times is embryonic in origin. TBP mRNA expression was sensitive to {alpha}-amanitin treatment beginning at 49 h post-hCG. CBP mRNA abundance was not reduced by {alpha}-amanitin treatment at any of the times analyzed, indicating a predominantly maternal origin throughout the period studied. Two non-TF mRNAs expressed as maternal transcripts were also included in the analysis. The rpL23 mRNA was expressed at a low abundance, and its expression was sensitive to {alpha}-amanitin treatment at both the mid and late 2-cell stages. The EF1{alpha} mRNA was abundantly expressed, and was partially reduced in abundance by {alpha}-amanitin treatment at the late 2-cell stage, indicating that the gene may become transcriptionally activated near the end of the 2-cell stage, but that a significant reservoir of maternal mRNA remains at that stage.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Effects of {alpha}-amanitin treatment on mRNA expression.a

The expression patterns of the TF mRNAs revealed differences with respect to maternal endowment to the embryo and embryonic transcription (Fig. 3). For example, the TBP mRNA appeared to be most abundant in GV-stage oocytes, declined somewhat in abundance during the 2-cell stage, and then increased very slowly in abundance beginning at the 4-cell stage following transcriptional activation of its gene. By contrast, the Sp1 mRNA was expressed at a relatively constant abundance until the 2-cell stage, when it began to increase in abundance after transcriptional activation of its gene. The CBP mRNA was most abundant in GV- and MII-stage oocytes, 1-cell and early 2-cell embryos; declined sharply in abundance during the 2-cell stage; and did not increase noticeably in abundance until the 16-cell or morula stage, consistent with the lack of an effect of {alpha}-amanitin during the early cleavage stages. The overall pattern of CBP mRNA expression resembled that of the TBP mRNA more than that of the Sp1 mRNA. The mTEAD-2 mRNA, as previously described [24], was expressed in GV- and MII-stage oocytes, and in 1-cell and 2-cell embryos, but underwent significant degradation during the 2-cell stage. A sharp increase in mTEAD-2 mRNA abundance occurs near the end of preimplantation development. The two non-TF mRNAs, rpL23 and EF1{alpha}, were both abundant in oocytes, declined in abundance during the 2-cell stage, and then were re-expressed at increasing abundances beginning at the 4- or 8-cell stages.

Polysome Association Pattern of the mTEAD-2 mRNA

The polyoma virus F101 enhancer first becomes able to enhance gene transcription at the 2-cell stage in developing mouse embryos [24]. This enhancer can be activated by members of the mTEAD family of TFs. The most predominant member of this TF family expressed in the 2-cell embryo is mTEAD, and it is expressed nearly to the exclusion of other family members [24]. The mTEAD-2 mRNA is expressed as a maternal mRNA that declines in abundance during development to the 2-cell stage. Because mTEAD-2 activity does not appear until the 2-cell stage [24], this mRNA appeared to be an excellent candidate for one that might be subject to stage-dependent translational recruitment. To test whether the mTEAD-2 mRNA is indeed recruited stage-specifically, we compared the distribution of this mRNA between polysomal and nonpolysomal fractions in MII oocytes, 1-cell embryos, and 2-cell embryos (Fig. 4). No significant difference in distribution among gradient fractions was observed between oocytes and 1-cell embryos. Two-cell embryos, however, exhibited a significant (P < 0.05) increase (~5-fold) in polysomal content for the mTEAD-2 mRNA.



View larger version (27K):
[in this window]
[in a new window]
  		FIG. 4. Distribution of specific mRNAs between sucrose density gradient zones for MII oocytes, 1-cell embryos, and 2-cell embryos. Sucrose density gradient fractionation was performed as described, and dot blots containing samples from upper nonpolysomal (T), intermediate (M), and bottom polysomal (B) fractions were hybridized to the indicated cDNA probes. Data are expressed as the fraction of total hybridization signal contained within the three gradient zones (mean ± SEM). For the 2-cell stage, some experiments employed {alpha}-amanitin treatment while others did not, as indicated

Polysome Association Pattern of Sp1 and TBP mRNAs

The strong inhibition of transcription of housekeeping genes by cycloheximide (CHX) treatment [20] indicates that TFs required for housekeeping gene transcription need to be synthesized during the 1-cell and/or 2-cell stages. Housekeeping genes generally have simple promoters and require general transcription factors (e.g., RNA polymerase, TBP) plus accessory factors and other widely acting TFs, like Sp1, for their transcription. Studies in 1-cell mouse embryos have reported an {alpha}-amanitin-independent increase in Sp1 protein abundance and significant effects of CHX on Sp1 and TBP nuclear protein abundance [58, 59]. These proteins, therefore, appeared as logical candidates with which to account for the effects of CHX on housekeeping gene transcription. To test whether the dependence of housekeeping gene transcription on protein synthesis is attributable to a stage-dependent recruitment of mRNAs encoding Sp1 and/or TBP, we compared the relative distribution of these mRNAs between polysomal and nonpolysomal fractions of oocytes, late 1-cell embryos, and 2-cell embryos (Fig. 4). We observed no significant increase in polysomal content for these mRNAs at either the 1-cell or 2-cell stages. Only a small fraction of TBP mRNA was contained within the polysomes at any stage, indicating that this mRNA is inefficiently translated in the egg and early embryo.

Polysome Association Pattern of the CBP mRNA

Transcriptional coactivators (e.g., p300/CBP) have been identified that promote transcription of a wide array of genes by interacting with a number of different TFs. Transcriptional activation occurs as a result of intrinsic histone acetyltransferase (HAT) activity or interaction with other factors that possess HAT activity [60–63]. Recruitment of these TFs with HAT activity to promoter complexes helps to destabilize chromatin, thus facilitating transcription. Because the early mouse embryo undergoes marked changes in histone acetylation status [64–66], including the elaboration of a unique nuclear domain that is enriched for acetylated histone isoforms [65], and because the CBP mRNA is expressed as a maternal transcript, we were interested to learn whether stage-specific recruitment of the CBP mRNA might occur, and thus contribute to embryonic genome activation (EGA) and the increased histone acetylation status by providing for an increase in abundance of CBP protein. There was no significant difference in distribution between polysomal and nonpolysomal fractions in oocytes, 1-cell embryos, and 2-cell embryos (Fig. 4).

Polysome Association Pattern of Other Maternal mRNAs

In order to compare the above TF mRNA polysome association patterns with the polysome association patterns of other maternal mRNAs that do not encode TFs, we examined the patterns of two other maternal mRNAs, EF1{alpha}, and rpL23 (Fig. 4). These two maternal mRNAs were both associated with polysomes during all three stages. A significant amount of EF1{alpha} mRNA was present within polysomes at all three stages, but its polysomal abundance decreased slightly during the 2-cell stage. The rpL23 mRNA was predominantly polysomal at all three stages, with nearly 90% of the mRNA residing within the polysomes in oocytes. There was a slight but significant (P < 0.05) decrease in rpL23 mRNA polysomal abundance at the 2-cell stage, but in comparison to other maternal mRNAs analyzed, the rpL23 mRNA appeared to be efficiently translated even in 2-cell-stage embryos.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Widespread transcriptional activation of the embryonic genome is a major event that must be regulated in the early embryo. Genome activation does not occur at a single discrete time, but rather comprises one or more early limited waves of genome activation followed by a major activation event. Thus, the regulation of embryonic genome activation may require a complex regulatory mechanism involving multiple levels of control, including changes in chromatin structure, post-translational modifications of maternal proteins, and the activation or synthesis of essential transcription factors (for review, see [3]).

Translational control of maternal mRNAs constitutes a major regulatory mechanism within early embryos. Translational control is accompanied in at least some cases by stage-specific mRNA deadenylation and exit from polyribosomes, or, alternatively, stage-specific polyadenylation and recruitment onto polyribosomes [43–45, 67–78]. Specific elements within the mRNAs themselves apparently contribute to the control of the temporal pattern of adenylation/deadenylation and entry into or exit from polyribosomes [57, 72–74, 78–80]. In some cases, transient polyadenylation also can occur [81].

The major genome activation event in the mouse embryo, which occurs during the 2-cell stage when the majority of housekeeping genes and many other genes are first transcribed, is dependent upon protein synthesis, consistent with the hypothesis that the recruitment of mRNAs encoding essential transcription factors provides one mechanism for controlling the time of the major genome activation event [20]. To test this hypothesis, we have examined the polyribosomal content of MII oocytes, 1-cell embryos, and 2-cell embryos for the presence of maternal mRNAs encoding different transcription factors. Our results reveal stage-specific and mRNA-specific differences in distribution between polysomal and nonpolysomal compartments for different maternal mRNAs during early embryogenesis, and, in particular, differences in the polysomal content of maternal mRNAs encoding transcription factors. Although the rate of synthesis of any given protein is affected by several parameters including the rate of translation initiation, number of ribosomes per transcript, and rate of polypeptide elongation, translational control in early embryos is often reflected as a shift of specific mRNAs into or out of the polyribosomal compartment. Accordingly, the differences in polyribosomal mRNA content reported here provide likely indicators of differences in the overall efficiency of translation of these maternal mRNAs in the oocyte and early embryo.

The mTEAD-2 mRNA exhibited a stage-dependent recruitment onto polysomes at the 2-cell stage. Recruitment of the mTEAD-2 mRNA occurs during the period in which the overall abundance of the mTEAD-2 mRNA is declining [24]. Thus, translation of the mTEAD-2 mRNA may be coupled to accelerated mRNA degradation, as has been observed for other mRNAs [82], or the mTEAD-2 mRNA may simply become less stable once it is mobilized. An alternative explanation for our data might be that the mTEAD-2 mRNA is not recruited onto polysomes, but rather that there is a loss from the cell of the nonpolyribosomal mTEAD-2 transcripts. This latter explanation, however, would not account for the earlier observation that mTEAD activity appears during the 2-cell stage, coincident with the apparent polysomal recruitment [24]. The simplest explanation for the available data is that the mTEAD-2 mRNA is translationally recruited, the mTEAD-2 activity then appears, and the mTEAD-2 mRNA is then degraded following recruitment. This interpretation is consistent with the hypothesis that regulated maternal mRNA recruitment can contribute to the control of the time of EGA by determining when specific TFs (e.g., mTEAD-2) appear.

Other TF mRNAs exhibit a continuous association with polysomes throughout the MII oocyte, 1-cell, and 2-cell stages. Of the TF mRNAs examined, this pattern is most evident for the Sp1 mRNA. A previous study documented an {alpha}-amanitin-independent increase in Sp1 protein content in 1-cell embryos [59]. Our data revealing a constitutive polysomal association of the Sp1 mRNA indicate that this increase in protein content may result from a change in protein stability or a change in rate of synthesis not related to mRNA recruitment per se (e.g., an increase in density of ribosomes on the Sp1 mRNA or the rate of peptide elongation). The effect of CHX on Sp1 nuclear protein content [58] and housekeeping gene transcription [20], together with the observed pattern of polysome association, indicates that continuous synthesis of Sp1 coupled to post-translational effects may contribute to widespread genome activation, especially activation of housekeeping genes.

We also observed that the Sp1 gene itself becomes transcriptionally active during the early phases of EGA (i.e., at the mid 2-cell stage), and maternally encoded Sp1 mRNA is largely eliminated by that time, in advance of the major transcriptional activation of housekeeping genes. From the mid 2-cell stage onward, Sp1 mRNA abundance increases steadily. The increase in Sp1 mRNA abundance during the 2-cell stage, and transcriptional activation of other TF genes with mRNAs regulated similarly, may thus be important for allowing the embryo to augment its supply of these TFs.

The mRNAs encoding TBP and CBP were poorly associated with polysomes throughout the period analyzed. Thus, it does not appear that translational recruitment of these mRNAs is likely to contribute to the control of the timing of EGA. Inefficient translation of these mRNAs in MII oocytes and early embryos obviously imposes a severe limit on the ability of the embryo to increase its supply of these TFs, and thus may cause these TFs to be present in limiting amounts in the early embryo. Treatment with CHX would further reduce the production of these TFs. Inhibition of protein synthesis with CHX produces a 4-fold decrease in nuclear TBP content [58], indicating that the maternally inherited TBP protein may undergo significant decay during the 1-cell and 2-cell stages. Thus, certain TFs like TBP and CBP may be especially limiting in the early embryo, and housekeeping gene transcription may be especially sensitive to a low abundance of these TFs, which could partially explain the effects of CHX on housekeeping gene transcription. Augmented expression of these TF mRNAs from the embryonic genome may therefore be a critical early need that must be satisfied in order to ensure viability. Consistent with this possibility, the TBP gene becomes transcriptionally active by the late 2-cell stage, initiating a gradual increase in TBP mRNA abundance.

An additional pattern of polysome association is exhibited by the rpL23 mRNA. This mRNA is predominantly polysomal throughout development, with > 80% of the message being on polysomes in MII oocytes and 1-cell embryos and > 50% in polysomes in 2-cell embryos. This pattern contrasts markedly with that observed for mRNAs like those encoding TBP and CBP. The predominantly polysomal association of this message indicates that the metabolic state of the egg and early embryo may favor efficient synthesis of ribosomal components, thus enhancing the embryonic protein synthesis capacity, which may in turn facilitate maternal mRNA recruitment. An enhancement of protein synthesis capacity in the embryo might thus facilitate EGA by facilitating maternal TF mRNA recruitment and also by allowing TFs to be translated immediately from early embryonic TF transcripts. It is also worth noting that the array of maternal mRNAs being translated may be affected by changes in general translation factor content, as well as translational control mechanisms that operate on a message-specific basis through cis elements (e.g., cytoplasmic polyadenylation elements) contained within certain messages, and this could also be part of the overall mechanism regulating EGA.

The results described are consistent with the hypothesis that both continuous synthesis of some TFs and regulated translational recruitment of maternal mRNAs encoding other TFs contribute to EGA and may affect its timing. Early transcription of TF genes may also contribute to sustained EGA as well. Very little is known about the array of genes activated at the 1-cell and 2-cell stages, the array of TF mRNAs contained within the egg cytoplasm, or the patterns of mRNA translation that exist in the mammalian embryo. The data presented here indicate that TF mRNAs are subject to different modes of regulation.


   ACKNOWLEDGMENTS
 
We thank Mel DePamphilis, Kotaro Kaneko, Elizabeth Moran, Peter Dallas, and Susannah Varmuza for generously providing probes used in the study.


   FOOTNOTES
 
First decision: 1 November 1999.

1 This work was supported by a Public Health Service grant (GM-56682). Back

2 Correspondence: Keith E. Latham, The Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, 3307 North Broad Street, Room 302, Philadelphia, PA 19140. FAX: 215 707 1454; klatham{at}unix.temple.edu Back

Accepted: November 17, 1999.

Received: September 27, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Latham KE. Epigenetic modification and imprinting of the mammalian genome during development. Curr Top Dev Biol 1999; 43:1–49.[Medline]
  2. Latham KE, Sapienza C. Localization of genes encoding egg modifiers of paternal genome function to mouse chromosomes one and two. Development 1998; 125:929–935.[Abstract]
  3. Latham KE. Mechanisms and control of embryonic genome activation in mammalian embryos. Int Rev Cytol 1999; 193:71–124.[Medline]
  4. Latham KE, Garrels JI, Solter D. Alterations in protein synthesis following transplantation of mouse 8-cell stage nuclei to enucleated 1-cell embryos. Dev Biol 1994; 163:341–350.[CrossRef][Medline]
  5. Latham KE, Solter D, Schultz RM. Acquisition of a transcriptionally permissive state during the one-cell stage of mouse embryogenesis. Dev Biol 1992; 149:457–462.[CrossRef][Medline]
  6. Aoki F, Worrad DM, Schultz RM. Regulation of transcriptional activity during the first and second cell cycles in the preimplantation mouse embryo. Dev Biol 1997; 181:296–307.[CrossRef][Medline]
  7. Clegg KB, Piko’ L. RNA synthesis and cytoplasmic polyadenylation in the one-cell mouse embryo. Nature 1982; 295:342–345.[CrossRef]
  8. Ram PT, Schultz RM. Reporter gene expression in G2 of the 1-cell mouse embryo. Dev Biol 1993; 156:552–556.[CrossRef][Medline]
  9. Thompson EM, Legouy E, Christians E, Renard J-P. Progressive maturation of chromatin structure regulates HSP70.1 gene expression in the preimplantation mouse embryo. Development 1995; 121:3425–3437.[Abstract]
  10. Vernet M, Bonnerot C, Briand P, Nicolas JF. Changes in permissiveness for the expression of microinjected DNA during the first cleavages of mouse embryos. Mech Dev 1992; 36:129–139.[CrossRef][Medline]
  11. Bensaude O, Babinet C, Morange M, Jacob F. Heat shock proteins, first major products of zygotic gene activity in mouse embryo. Nature 1983; 305:331–333.[CrossRef][Medline]
  12. Conover JC, Temeles GL, Zimmerman JW, Burke B, Schultz RM. Stage-specific expression of a family of proteins that are major products of zygotic gene activation in the mouse embryo. Dev Biol 1991; 144:393–404.
  13. Flach G, Johnson MH, Braude PR, Taylor RAS. Bolton VN. The transition from maternal to embryonic control in the 2-cell mouse embryo. EMBO J 1982; 1:681–686.[Medline]
  14. Latham KE, Garrels J, Chang C, Solter D. Quantitative analysis of protein synthesis in preimplantation mouse embryos: extensive reprogramming at the one- and two-cell stages. Development 1991; 112:921–932.[Abstract]
  15. Latham KE, Garrels JI, Chang C, Solter D. Analysis of protein synthesis in mouse embryos: construction of a high-resolution, two-dimensional gel protein database. Appl Theor Electrophoresis 1992; 2:163–170.[Medline]
  16. Sawicki JA, Magnuson T, Epstein CJ. Evidence for expression of the paternal genome in the two-cell mouse embryo. Nature 1981; 294:450–451.[CrossRef][Medline]
  17. Telford NA, Watson AJ, Schultz GA. Transitions from maternal to embryonic control in early mammalian development: a comparison of several species. Mol Reprod Dev 1990; 26:90–100.[CrossRef][Medline]
  18. Memili E, First NL. Developmental changes in RNA polymerase II in bovine oocytes, early embryos, and effect of alpha-amanitin on embryo development. Mol Reprod Dev 1998; 51:381–389.[CrossRef][Medline]
  19. Taylor DM, Ray PF, Ao A, Winston RM, Handyside AH. Paternal transcripts for glucose-6-phosphate dehydrogenase and adenosine deaminase are first detectable in the human preimplantation embryo at the three- to four-cell stage. Mol Reprod Dev 1997; 48:442–448.[CrossRef][Medline]
  20. Wang Q, Latham KE. A role for protein synthesis during embryonic genome activation in mice. Mol Reprod Dev 1997; 47:265–270.[CrossRef][Medline]
  21. Ho Y, Wigglesworth K, Eppig JJ, Schultz RM. Preimplantation development of mouse embryos in KSOM: augmentation by amino acids and analysis of gene expression. Mol Reprod Dev 1995; 41:232–238.[CrossRef][Medline]
  22. Rambhatla L, Patel B, Dhanasekaran N, Latham KE. Analysis of G protein {alpha} subunit mRNA abundance in preimplantation mouse embryos using a rapid, quantitative RT-PCR approach. Mol Reprod Dev 1995; 41:314–324.[CrossRef][Medline]
  23. Persengiev SP, Saffer JD, Kilpatrick DL. An alternatively spliced form of the transcription factor Sp1 containing only a single glutamine-rich transactivation domain. Proc Natl Acad Sci USA 1995; 92:9107–9111.[Abstract/Free Full Text]
  24. Kaneko KJ, Cullinan EB, Latham KE, DePamphilis ML. Transcription factor mTEAD2 is selectively expressed at the beginning of zygotic gene expression in the mouse. Development 1997; 124:1963–1973.[Abstract]
  25. Varmuza S, Tate P. Isolation of epiblast-specific cDNA clones by differential hybridization with polymerase chain reaction-amplified probes derived from single embryos. Mol Reprod Dev 1992; 32:339–348.[CrossRef][Medline]
  26. Manejwala FM, Logan CY, Schultz RM. Regulation of hsp70 mRNA levels during oocyte maturation and zygotic gene activation in the mouse. Dev Biol 1991; 144:301–308.[CrossRef][Medline]
  27. Singer-Sam J, Chapman V, LeBon JM, Riggs AD. Parental imprinting studied by allele-specific primer extension after PCR: paternal X chromosome-linked genes are transcribed prior to preferential paternal X chromosome inactivation. PNAS USA 1992; 89:10469–10473.[Abstract/Free Full Text]
  28. Bergeron L, Perez GI, McDonald G, Shi L, Sun Y, Jurisicova A, Varmuza S, Latham K, Flaws JA, Salter J, Hara H, Moskowitz MA, Li E, Greenberg AH, Tilly JL, Yuan J. Defects in regulation of apoptosis in Caspase-2 deficient mice. Genes Dev 1998; 12:1303–1317.
  29. Domachenko A, Latham KE, Hatton KS. Expression of myc-family, myc-interacting, and myc target genes during preimplantation mouse development. Mol Reprod Dev 1997; 47:57–65.[CrossRef][Medline]
  30. Jurisicova A, Latham KE, Casper RF, Varmuza SL. Expression and regulation of genes associated with cell death during murine preimplantation embryo development. Mol Reprod Dev 1998; 51:243–253.[CrossRef][Medline]
  31. Latham KE, Rambhatla L. Expression of X linked genes in androgenetic, gynogenetic, and normal mouse preimplantation embryos. Dev Genet 1995; 17:212–222.[CrossRef][Medline]
  32. Latham KE, Litvin J, Orth J, Mettus R, Reddy EP. Temporal patterns of A-myb and B-myb gene expression during testis development. Oncogene 1996; 13:1161–1168.[Medline]
  33. Latham KE, Rambhatla L, Hayashizaki Y, Chapman VM. Stage-specific induction and regulation by genomic imprinting of the mouse U2afbp-rs gene during preimplantation development. Dev Biol 1995; 168:670–676.[CrossRef][Medline]
  34. Latham KE, Kutyna K, Wang Q. Genetic variation in trophectoderm function in parthenogenetic mouse embryos. Dev Genet 1999; 24:329–335.[CrossRef][Medline]
  35. Mann M, Latham KE, Varmuza S. Identification of genes showing altered expression in preimplantation and early postimplantation parthenogenetic embryos. Dev Genet 1995; 17:223–232.[CrossRef][Medline]
  36. Peterson K, Wang G. Horsley D, Richardson JC, Sapienza C, Latham KE, Singh P. The M31 gene has a complex developmentally regulated expression profile and may encode alternative protein products that possess diverse subcellular localisation patterns. J Exp Zool 1998; 280:288–303.[CrossRef][Medline]
  37. Rossant J, Guillemot F, Tanaka M, Latham K, Gertenstein M, Nagy A. Mash2 is expressed in oogenesis and preimplantation development but is not required for blastocyst formation. Mech Dev 1998; 73:183–191.[CrossRef][Medline]
  38. Brady G, Iscove N. Construction of cDNA libraries from single cells. Methods Enzymol 1993; 225:611–623.[Medline]
  39. Brady G, Billia F, Knox J, Hoang T, Kirsch IR, Voura EB, Hawley RG, Cumming R, Buchwald M, Siminovitch K. Analysis of gene expression in a complex differentiation hierarchy by global amplification of cDNA from single cells. Curr Biol 1995; 5:909–922.[CrossRef][Medline]
  40. Cano-Gauci DF, Lualdi JC, Ouellette AJ, Brady G, Iscove NN, Buick RN. In vitro cDNA amplification from individual intestinal crypts: a novel approach to the study of differential gene expression along the crypt-villus axis. Exp Cell Res 1993; 208:344–349.[CrossRef][Medline]
  41. Weaver DL, Nunez C, Brunet C, Bostock V, Brady G. Single-cell RT-PCR cDNA subtraction. Methods Mol Biol 1999; 97:601–609.[Medline]
  42. Huarte J, Belin D, Vassalli JD. Plasminogen activator in mouse and rat oocytes: induction during meiotic maturation. Cell 1985; 43:551–558.[CrossRef][Medline]
  43. Huarte J, Belin D, Vassalli A, Strickland S, Vassalli J-D. Meiotic maturation of mouse oocytes triggers the translation and polyadenylation of dormant tissue-type plasminogen activator mRNA. Genes Dev 1987; 1:1201–1211.[Abstract/Free Full Text]
  44. Vassalli J-D, Huarte J, Belin D, Gubler P, Vassalli A, O'Connell ML, Parton LA, Rickles RJ, Strickland S. Regulated polyadenylation controls mRNA translation during meiotic maturation of mouse oocytes. Genes Dev 1989; 3:2163–2171.[Abstract/Free Full Text]
  45. Paynton BV, Rempel R, Bachvarova R. Changes in state of adenylation and time course of degradation of maternal mRNAs during oocyte maturation and early embryonic development in the mouse. Dev Biol 1988; 129:304–314.[CrossRef][Medline]
  46. Williams CJ, Schultz RM, Kopf GS. G protein gene expression during mouse oocyte growth and maturation, and preimplantation embryo development. Mol Reprod Dev 1996; 44:315–323.[CrossRef][Medline]
  47. Dooley TP, Miranda M, Jones NC, DePamphilis ML. Transactivation of the adenovirus EIIa promoter in the absence of E1a protein is restricted to mouse oocytes and preimplantation embryos. Development 1989; 107:945–956.[Abstract/Free Full Text]
  48. Paria BC, Dey S. Antisense c-myc effects on preimplantation mouse embryo development. Proc Natl Acad Sci USA 1992; 89:10051–10055.[Abstract/Free Full Text]
  49. Hatada I, Kazunori K, Yamaoka T, Wang X, Arai Y, Hashido K, Ohishi S, Masuda J, Ogata J, Mukai T. Allele-specific methylation and expression of an imprinted U2af1-rs1 (SP2) gene. Nucleic Acids Res 1995; 23:36–41.[Abstract/Free Full Text]
  50. Exley GE, Tang C, McElhinny AS, Warner CM. Expression of caspase and bcl-2 apoptotic family members in mouse preimplantation embryos. Biol Reprod 1999; 61:231–239.[Abstract/Free Full Text]
  51. Warner CM, Exley GE, McElhinny AS, Tang C. Genetic regulation of preimplantation mouse embryo survival. J Exp Zool 1998; 282:272–279.[CrossRef][Medline]
  52. Watson AJ, Pape C, Emanual JR, Levenson R, Kidder GM. Expression of Na+,K+-ATPase {alpha} and ß subunit genes during preimplantation development of the mouse. Dev Genet 1990; 11:41–48.[CrossRef][Medline]
  53. Kidder GM, Watson AJ. Gene expression required for blastocoel formation in the mouse. In: Heyner S, Wiley LM (eds.), Early Embryo Development and Paracrine Relationships. New York: Wiley-Liss; 1990: 97–107.
  54. Daniels R, Zuccotti M, Kinis T, Serhal P, Monk M. XIST expression in human oocytes and preimplantation embryos. Am J Hum Genet 1997; 61:33–39.[Medline]
  55. Goto T, Christians A, Monk M. Expression of an Xist promoter-luciferase construct during spermatogenesis and in preimplantation embryos: regulation by DNA methylation. Mol Reprod Dev 1998; 49:356–367.[CrossRef][Medline]
  56. Kay GF, Penny GD, Patel D, Ashworth A, Brockdorff N, Rastan S. Expression of Xist during mouse development suggests a role in the initiation of X chromosome inactivation. Cell 1993; 72:171–182.[CrossRef][Medline]
  57. Huarte J, Stutz A, O'Connell ML, Gubler P, Belin D, Darrow AL, Strickland S, Vassalli JD. Transient translational silencing by reversible mRNA deadenylation. Cell 1992; 69:1021–1030.[CrossRef][Medline]
  58. Worrad DM, Ram PT, Schultz RM. Regulation of gene expression in the mouse oocyte and early preimplantation embryo: developmental changes in Sp1 and TATA box binding protein, TBP. Development 1994; 120:2347–2357.[Abstract]
  59. Worrad DM, Schultz RM. Regulation of gene expression in the preimplantation mouse embryo: temporal and spatial patterns of expression of the transcription factor Sp1. Mol Reprod Dev 1997; 46:268–277.[CrossRef][Medline]
  60. Bannister AJ, Kouzarides T. The CBP co-activator is a histone acetyltransferase. Nature 1996; 384:641–643.[CrossRef][Medline]
  61. Ogryzko VV, Schiltz RL, Russanova V, Howard BH, Nakatani Y. The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell 1996; 87:953–959.[CrossRef][Medline]
  62. Utley RT, Ikeda K, Grant PA, Cote J, Steger DJ, Eberharter A, John S, Workman JL. Transcriptional activators direct histone acetyltransferase complexes to nucleosomes. Nature 1998; 394:498–502.[CrossRef][Medline]
  63. Wolffe AP, Hayes JJ. Chromatin disruption and modification. Nucleic Acids Res 1999; 27:711–720.[Abstract/Free Full Text]
  64. Adenot PG, Mercier Y, Renard J-P, Thompson EM. Differential H4 acetylation of paternal and maternal chromatin precedes DNA replication and differential transcriptional activity in pronuclei of 1-cell mouse embryos. Development 1997; 124:4615–4625.[Abstract]
  65. Stein P, Worrad DM, Belyaev N, Turner BM, Schultz RM. Stage-dependent redistributions of acetylated histones in nuclei of the early preimplantation mouse embryo. Mol Reprod Dev 1997; 47:421–429.[CrossRef][Medline]
  66. Worrad DM, Turner BM, Schultz RM. Temporally restricted spatial localization of acetylated isoforms of histone H4 and RNA polymerase II in the 2-cell mouse embryo. Development 1995; 121:2949–2959.[Abstract]
  67. Bachvarova R, De Leon V. Polyadenylated RNA of mouse ova and loss of maternal RNA in early development. Dev Biol 1980; 74:1–8.[CrossRef][Medline]
  68. Bachvarova R, De Leon V, Johnson A, Kaplan G, Paynton BV. Changes in total RNA, polyadenylated RNA, and actin mRNA during meiotic maturation of mouse oocytes. Dev Biol 1985; 108:325–331.[CrossRef][Medline]
  69. Brevini-Gandolfi TA, Favetta LA, Mauri L, Luciano AM, Cillo F, Gandolfi F. Changes in poly(A) tail length of maternal transcripts during in vitro maturation of bovine oocytes and their relation with developmental competence. Mol Reprod Dev 1999; 52:427–333.[CrossRef][Medline]
  70. de Moor CH, Richter JD. The Mos pathway regulates cytoplasmic polyadenylation in Xenopus oocytes. Mol Cell Biol 1997; 17:6419–6426.[Abstract]
  71. Hwang SY, Oh B, Fuchtbauer A, Fuchtbauer EM, Johnson KR, Solter D, Knowles BB. Maid: a maternally transcribed novel gene encoding a potential negative regulator of bHLH proteins in the mouse egg and zygote. Dev Dyn 1997; 209:217–226.[CrossRef][Medline]
  72. Oh B, Hwang SY, Solter D, Knowles BB. Spindlin, a major maternal transcript expressed in the mouse during the transition from oocyte to embryo. Development 1997; 124:493–503.[Abstract]
  73. Paris J, Swenson K, Piwnica-Worms H, Richter JD. Maturation-specific polyadenylation: in vitro activation by p34cdc2 and phosphorylation of a 58-kD CPE binding protein. Genes Dev 1991; 5:1697–1708.[Abstract/Free Full Text]
  74. Sheets MD, Fox CA, Hunt T, Vande Woude G, Wickens M. The 3' untranslated regions of c-mos and cyclin mRNAs stimulate translation by regulating cytoplasmic polyadenylation. Genes Dev 8:926–938.
  75. Simon R, Tassan J-P, Richter JD. Translational control by poly(A) elongation during Xenopus development: differential repression and enhancement by a novel cytoplasmic polyadenylatiohn element. Genes Dev 1992; 6:2580–2591.[Abstract/Free Full Text]
  76. Simon R, Wu L, Richter JD. Cytoplasmic polyadenylation of activin receptor mRNA and the control of pattern formation in Xenopus development. Dev Biol 1996; 179:239–250.[CrossRef][Medline]
  77. Verotti AC, Thompson SR, Wreden C, Strickland S, Wickens M. Evolutionary conservation of sequence elements controlling cytoplasmic localization. Proc Natl Acad Sci USA 1996; 93:9027–9032.[Abstract/Free Full Text]
  78. West MF, Verrotti AC, Salles FJ, Tsirka SE, Strickland S. Isolation and characterization of two novel cytoplasmically polyadenylated oocyte-specific mouse maternal mRNAs. Dev Biol 1996; 175:132–141.[CrossRef][Medline]
  79. Fox CA, Wickens M. Poly(A) removal during oocyte maturation: a default reaction selectively prevented by specific sequences in the 3' UTR of certain maternal mRNAs. Genes Dev 1990; 4:2287–2298.[Abstract/Free Full Text]
  80. Varnum SM, Wormington WM. Deadenylation of maternal mRNAs during Xenopus oocyte maturation does not require specific cis-sequences: a default mechanism for translational control. Genes Dev 1990; 4:2278–2286.[Abstract/Free Full Text]
  81. Temeles GL, Schultz RM. Transient polyadenylation of a maternal mRNA following fertilization of mouse eggs. J Reprod Fertil 1997; 109:223–228.[Abstract]
  82. Winstall E, Gamache M, Raymond V. Rapid mRNA degradation mediated by the c-fos 3' AU-rich element and that mediated by the granulocyte-macrophage colony-stimulating factor 3' AU-rich element occur through similar polysome-associated mechanisms. Mol Cell Biol 1995; 15:3796–3804.[Abstract]



This article has been cited by other articles:


Home page
 Biol. Reprod.Home page
S.-I. Kageyama, M. Nagata, and F. Aoki
Isolation of Nascent Messenger RNA from Mouse Preimplantation Embryos
Biol Reprod, December 1, 2004; 71(6): 1948 - 1955.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
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]

Home page
 Biol. Reprod.Home page
P. Zheng, B. Patel, M. McMenamin, S. E. Reddy, A. M. Paprocki, R. D. Schramm, and K. E. Latham
The Primate Embryo Gene Expression Resource: A Novel Resource to Facilitate Rapid Analysis of Gene Expression Patterns in Non-Human Primate Oocytes