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Biology of Reproduction 65, 748-754 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Role of Protein Synthesis in the Development of a Transcriptionally Permissive State in One-Cell Stage Mouse Embryos1

Qingxue Wanga, Young Gie Chunga, Wilhelmine N. deVriesc, Marina Struwed, and Keith E. Latham2,a,b

a Fels Institute for Cancer Research and Molecular Biology, b Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140 c The Jackson Laboratory, Bar Harbor, Maine 04609 d The Max-Planck Institute for Immunobiology, 79011 Freiburg, Germany

ABSTRACT

The time of onset of gene transcription in the mouse embryo is temporally regulated. A prominent feature of this regulation is a change during the one-cell stage from a transcriptionally nonpermissive state to a transcriptionally permissive state. During the early one-cell stage, the cytoplasm is either inadequate or suppressive for nuclear gene transcription, but by the late one-cell stage, the cytoplasm acquires the ability to support gene transcription either in endogenous nuclei or exogenous nuclei introduced microsurgically. We have investigated the role of protein synthesis in this cytoplasmic transition. Nuclei from two-cell stage embryos treated with {alpha}-amanitin were used to evaluate the transcriptional permissiveness of late one-cell stage cytoplasm, as indicated by the production of transcripts from four genes that are specifically transcribed at elevated rates during the two-cell stage. Two of these genes were transcribed following nuclear transfer to late one-cell stage cytoplasm, and two were not transcribed. Treatment of the recipient cytoplasm with cycloheximide to inhibit protein synthesis from the early to the late one-cell stage inhibited the transcription of the two genes that were transcribed in the untreated, late one-cell stage recipients. These results indicate that acquisition of the transcriptionally permissive state during the one-cell stage is facilitated by protein synthesis, and that the transcriptional permissiveness in the late one-cell stage cytoplasm is limited to certain genes.

developmental biology, gene regulation

INTRODUCTION

Fertilization results in the union of two dissimilar haploid genomes from two different types of cells within a common cytoplasm. These haploid genomes must then be reprogrammed to function as a totipotent embryonic genome. Once formed, the early mammalian genome is initially transcriptionally silent. The initial period of transcriptional silence may serve a protective function by restricting the access of certain modifying factors to the embryonic genome [1]. Data from nuclear transplantation studies indicate that gene transcription during the early period of nuclear reprogramming may be deleterious. For example, transplantation of transcriptionally active eight-cell stage nuclei to one-cell stage cytoplasm can lead to irreversible repression of certain genes, most likely through inappropriate epigenetic modifications involving, perhaps, changes in chromatin structure [1]. Given the potentially deleterious effects of premature gene transcription, it is reasonable to expect that the newly fertilized embryo will manifest regulatory mechanisms that delay transcriptional activation of the embryonic genome and, perhaps, limit the array of genes that are first transcribed.

The mammalian embryonic genome undergoes transcriptional activation as early as the late one-cell stage [2–11]. Subsequently, an increase in gene transcription activity occurs in a stepwise manner, so that although some genes are activated during the late one-cell stage, many other genes become activated at later cleavage stages [12–31]. The stepwise nature of embryonic genome activation is consistent with a model in which multiple regulatory mechanisms control the activation of different subsets of genes during the different waves of transcriptional activation.

In the mouse embryo, one of the earliest-acting mechanisms controlling transcription is a stage-dependent change in the ability of one-cell stage cytoplasm to support gene transcription in the nucleus. The early one-cell stage cytoplasm does not support expression of the two-cell stage-specific, 70-kDa transcription-requiring complex (TRC) when transcriptionally competent, {alpha}-amanitin-treated (RNA polymerase II-inhibited), two-cell stage nuclei are introduced by nuclear transplantation. By contrast, TRC expression occurs when nuclei are transplanted to late one-cell stage cytoplasm [32]. This transition to a transcriptionally permissive state at the late one-cell stage is consistent with the ability of late one-cell stage embryos to transcribe exogenous reporter genes, transgenes, and endogenous genes [3–7, 9]. The difference between early and late one-cell stage cytoplasm in the ability to support transcription in transplanted two-cell stage nuclei indicates that the early one-cell stage cytoplasm either 1) contains one or more factors that can enter the donor nuclei and inhibit transcription, 2) lacks a sufficient amount of active RNA polymerase II to restore transcription to the {alpha}-amanitin-inhibited donor nuclei, or 3) lacks a sufficient amount of other transcription factors in an active state to support gene transcription. Similar restrictions would likely apply to endogenous gene transcription (i.e., in one-cell stage nuclei). The late one-cell stage cytoplasm, by contrast, either lacks the inhibitors or has acquired a sufficient amount of active RNA polymerase II or other transcription factors to support transcription in the transplanted nuclei or in endogenous nuclei.

To our knowledge, it has not been determined whether the transcriptionally permissive state that arises during the one-cell stage is limited with respect to the array of genes that can be transcribed, or whether the transition to this permissive state requires novel proteins to be produced or, instead, is directed solely by maternal proteins in the cytoplasm. To address these questions, we examined the expression of four different two-cell stage-specific transcripts (i.e., mRNAs showing transient, transcription-dependent elevations in abundance during the two-cell stage) following transfer of two-cell stage nuclei to late one-cell stage cytoplasm. Our data indicate that the array of genes that can be transcribed during the late one-cell stage is limited, and that gene transcription during the late one-cell stage is dependent on protein synthesis.

MATERIALS AND METHODS

Embryos and Embryo Culture

The temporal expression patterns and sensitivity to {alpha}-amanitin treatment for the genes assayed were obtained by cDNA dot-blot hybridization analyses employing embryos from superovulated (B6D2)F1 females as described by Rambhatla et al. [27]. For nuclear transplantation experiments, embryos were obtained from superovulated Swiss albino females (Harlan, Indianapolis, IN). Females were superovulated by injection of 5 IU of equine chorionic gonadotropin, followed 48 h later by 5 IU of human chorionic gonadotropin (hCG), and then mated to (B6D2)F1 males (Taconic Farms, Germantown, NY) as described previously [32]. Embryos were cultured in CZB medium [33] under an atmosphere of 5% CO2, 5% O2, and 90% N2, as in our previous study [32]. All studies adhered to procedures consistent with the National Research Council Guide for the Care and Use of Laboratory Animals.

Nuclear Transplantation

Nuclear transplantation was performed using inactivated Sendai virus to mediate cell fusion as described previously [32], except that karyoplast transfers were performed in Hepes-buffered CZB medium [34]. Donor embryos were isolated at approximately 20 h post-hCG and cultured overnight from the mid one-cell stage onward in the presence of 24 µg/ml of {alpha}-amanitin and 5 µg/ml of cytochalasin B (Fig. 1). The cytochalasin B was employed to prevent cleavage and, thereby, allow transplantation of both donor two-cell stage nuclei in a single manipulation, as performed previously [32]. Because cytochalasin treatment does not prevent DNA replication, this strategy allows the equivalent DNA content of a control two-cell stage embryo to be introduced into the nuclear transplant embryos [32]. The {alpha}-amanitin treatment inactivates endogenous RNA polymerase II, preventing the accumulation of mRNAs encoded by genes that have been developmentally programmed for expression and, thus, the transfer of such embryonically transcribed mRNAs during microsurgery [32]. Recipient embryos were isolated at 20 h post-hCG and cultured in the presence or absence of cycloheximide (20 µg/ml, a concentration previously demonstrated to inhibit protein synthesis completely [35]). Nuclei were introduced at approximately 26 h post-hCG, and the embryos were cultured for 4 h before lysis. Care was taken to transfer as little cytoplasm (estimated as <=0.2%) as possible with the nuclei. Cycloheximide treatment was continued during the 4-h incubation following nuclear transfer to cycloheximide-treated recipients. Embryos were lysed 4 h after microsurgery and were still at the one-cell stage. Lysis was performed at 4 h after nuclear transfer, because our previous data for TRC synthesis were obtained with a 4-h incubation before labeling with 35S-methionine [32]. Moreover, it was imperative not to allow the incubation to extend beyond the time when the recipients would be chronologically at the one-cell stage. Enucleated cytoplasts (untreated or treated with cycloheximide) were cultured in parallel as controls. As additional controls, two-cell stage embryos, either untreated or treated overnight as described above with {alpha}-amanitin (i.e., from mid one-cell stage onward), were lysed at approximately the same time as when nuclear transplantation was performed.



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  		FIG. 1. Design of nuclear transfer experiments. Transcriptionally competent two-cell stage embryos pretreated with {alpha}-amanitin and cytochalasin B were used as nuclear donors. The enucleated late one-cell embryos, either untreated or treated with cycloheximide (CHX), were used as recipients. Embryos were cultured for 4 h after nuclear transfer and then processed for RT-PCR analysis. hphCG, Hours post-hCG

Complementary DNA Probes

The cDNA fragment for glucose-6-phosphate dehydrogenase (G6pd) mRNA was 611 base pairs (bp) in length [24]. The 1.8-kilobase (kb) NotI/EcoRV U2afbp-rs cDNA fragment was isolated from the D2NV2 clone [36, 37]. The 1.2-kb EcoRI cDNA fragment corresponding to the 3' end of the eIF-1A mRNA was recovered from a plasmid clone [38]. The cDNA clone for ERV-L consisted of a 1982-bp, partial cDNA clone corresponding to nucleotides 3484–5466 of the published viral sequence [39]. A full-length cDNA (designated L2) representing a gene family denoted as Alberich was used. The ERV-L and Alberich cDNAs were initially identified in a screen for transcripts that are transiently elevated in abundance at the two-cell stage (S.Y. Huang, W. N. DeVries, M. Struwe, B. Knowles, personal communication).

Reverse Transcription-Polymerase Chain Reaction Assay

The abundances of specific mRNAs were measured using the Quantitative Amplification and Dot Blotting (QADB) method for quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis [27, 40]. Briefly, zona-free embryos were lysed in RT buffer supplemented with NP-40, RNase inhibitors, nucleotides, and oligo(dT) [27, 40]. After heat denaturation and annealing, RT-PCR was performed as described previously [27, 40] to amplify quantitatively the 3' terminal portions of the entire mRNA population. Quantitation of specific mRNAs was achieved by dot blotting and cDNA hybridization as described previously [27, 40]. The sensitivity and reliability of the QADB assay have been extensively documented [41]. The QADB method is applicable to small amounts of material (even single embryos), provides the ability to quantify expression of a large number of mRNAs, and can provide estimates of actual mRNA abundance [27, 40]. These properties make the QADB method ideal for examining mRNA abundances in nuclear transplant embryos. The QADB method is fully quantitative and produces hybridization signals that are linear over at least three orders of magnitude (R2 = 0.992), extending to a very low mRNA abundance and with excellent reproducibility [41]. The QADB method also exhibits excellent reproducibility between experiments with respect to qualitative patterns of mRNA expression and quantitative estimates of mRNA abundance [24, 27]. For the determination of temporal patterns of expression, the data obtained by the QADB method were expressed in units of counts per minute bound (cpm bound). Between three and six samples of 8–10 embryos each were collected for each stage analyzed. For the nuclear transfer experiments, between 4 and 10 samples of 6–10 embryos each were obtained for each type of embryo analyzed. To improve sensitivity and precision of measurements for low-abundance signals, three measurements were obtained for each sample in the nuclear transfer experiments, and the average values were used for further calculations. The statistical significance of differences in the means was evaluated using the Student t-test.

RESULTS AND DISCUSSION

In our previous studies, acquisition of the transcriptionally permissive state was detected by synthesis of the 70-kDa TRC protein complex [32]. The present study was designed to evaluate the effects of inhibiting protein synthesis on gene transcription in the transplanted donor nuclei. Because synthesis of the 70-kDa protein complex obviously cannot be employed when protein synthesis is inhibited, we evaluated transcription following nuclear transplantation in the present study by assaying for the expression of four mRNAs encoded by genes that are transcriptionally induced transiently during the two-cell stage in a manner similar to that observed for the TRC. These included the mRNAs encoding the putative splicing factor U2afbp-rs, the translation factor eIF-1A, the endogenous murine retrovirus ERV-L, and a member of a multigene family informally referred to as Alberich (B. Knowles, personal communication). All four genes are transiently induced during the two-cell stage (Fig. 2A) [36, 38]. Expression of all four mRNAs was greatly inhibited by {alpha}-amanitin treatment at the two-cell stage (Table 1), indicating very little maternal mRNA contribution at that stage. Because only a very small amount of cytoplasm is transplanted with the donor nuclei, the amount of mRNA expected to be transplanted with the nuclei from {alpha}-amanitin-treated donor embryos is, thus, negligible. Expression of all four mRNAs during the late one-cell stage was also very low compared with that during the two-cell stage (Table 1 and Fig. 2A). The maternally derived G6pd mRNA (Fig. 2A) [24] was assayed as a control.



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  		FIG. 2. Temporal expression patterns of mRNAs during preimplantation development. A) Embryos were collected at different times after hCG injection, as indicated at the bottom of the graph. The corresponding developmental stages, including germinal vesicle-intact oocyte (GVOOC), egg, one-cell, two-cell, four-cell, eight-cell, morula, and blastocyst, are indicated above the graph. Hybridization signals were normalized to the amount of DNA bound to each dot, as described by Rambhatla et al. [27] and Latham et al. [40], and then expressed in units of bound counts per minute (cpm) after conversion of PhosphorImager data using a dot with a known quantity of 32P for calibration. Bars indicate the mean value (± SEM) obtained for each embryo type or stage indicated. Data for U2afbp-rs and G6pd are reproduced from Latham et al. [36] and from Latham and Rambhatla [24], respectively, and are presented here for comparative purposes. B) A representative dot blot illustrating enhanced expression of Alberich mRNA at the two-cell stage. The dots grouped under the drawn lines correspond to samples from: 1, 39-h post-hCG untreated embryos; 2, 39-h post-hCG {alpha}-amanitin-treated embryos; 3, 49-h post-hCG untreated embryos; 4, 49-h post-hCG {alpha}-amanitin-treated embryos; and 5, two-cell embryos isolated from the oviduct and lysed without culturing. Dots preceding group 1 correspond to samples ranging from, left to right and top to bottom, germinal vesicle-stage oocytes, unfertilized eggs, one-cell stage (18, 22, and 29 h post-hCG), and early two-cell stage (32 h post-hCG), and dots following group 5 correspond to samples of four-cell stage through blastocyst embryos and inner cell mass (57–118 h post-hCG; see A)


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  		TABLE 1. Effects of {{alpha}}-amanitin treatment on mRNA expression.a

We wished to determine whether the late one-cell stage cytoplasm becomes transcriptionally permissive for genes other than the TRC gene(s) and, if so, whether protein synthesis is required. If the four transiently induced genes respond to the late one-cell stage cytoplasm in a manner similar to the 70-kDa TRC protein complex gene(s), then we would expect to observe a statistically significant increase in the abundance of these mRNAs in nuclear transplant embryos relative to their abundance in enucleated cytoplasts. In our previous study, the TRC protein was expressed approximately 40% as abundantly in the nuclear transplant embryos as in normal two-cell stage embryos [32]. It should be noted, however, that we have no reason to expect that the amount of any given mRNA accumulated following nuclear transplantation should equal the amount present in normal two-cell stage embryos. The accumulation of mRNA encoded by the donor nuclei requires that the nucleus acquire functional RNA polymerase from the recipient cytoplasm, that the target genes be activated, and that the mRNAs accumulate to detectable quantities within a period of 4 h. Thus, the expression of a given mRNA at an abundance significantly greater in nuclear transplant embryos compared with that in enucleated controls is the expected indicator of transcriptional activity.

Transplantation of {alpha}-amanitin-treated donor nuclei to enucleated, late one-cell stage cytoplasm resulted in an increased expression of the U2afbp-rs mRNA (Fig. 3). The mean U2afbp-rs mRNA abundance in late one-cell stage nuclear transplant embryos was significantly greater than that in enucleated one-cell stage cytoplasts (1.3-fold, P < 0.01). Treatment of one-cell stage cytoplasm with cycloheximide from 20 to 26 h post-hCG reduced U2afbp-rs mRNA expression in nuclear transplant embryos by approximately twofold compared with that in untreated nuclear transplant embryos (P < 1 x 10-6). Moreover, expression in cycloheximide-treated nuclear transplant embryos did not differ significantly from cycloheximide-treated enucleated cytoplasts, indicating that transcription did not occur in the cycloheximide-treated nuclear transfer embryos. These data indicate that the U2afbp-rs gene was transcribed in the nuclear transplant embryos, and that this transcription was inhibited by cycloheximide treatment.



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  		FIG. 3. Expression of mRNAs in nuclear transfer embryos and control embryos. Data represent the mean hybridization signals (± SEM) expressed as a percentage of maximum signals. Inset graphs show details of expression for the first six bars of the parent graph. Asterisks denote instances in which mean expression values for nuclear transplant embryos differ significantly from the values for their enucleated controls

The ERV-L mRNA was expressed 2.6-fold more abundantly in the late one-cell stage nuclear transplant embryos than in enucleated one-cell stage cytoplasts (P < 0.02). In contrast to the U2afbp-rs mRNA, the ERV-L mRNA was expressed at a very low abundance in nuclear transplant embryos compared with that in two-cell stage embryos. Based on our earlier examination regarding the sensitivity of the QADB assay [41], however, the intensity of hybridization signals obtained with nuclear transplant embryos were well within the linear range of the assay. Cycloheximide treatment inhibited ERV-L mRNA expression by 2.8-fold (P < 0.001) comparing treated and untreated nuclear transplant embryos. Cycloheximide-treated nuclear transplant embryos and cycloheximide-treated enucleated cytoplasts did not differ significantly in expression (P > 0.05). These data indicate that the ERV-L gene was transcribed at a low level in nuclear transplant embryos, and that this transcription was also sensitive to cycloheximide treatment.

Expression of neither the eIF-1A nor the Alberich gene was significantly elevated in late one-cell nuclear transplant embryos compared with enucleated one-cell controls. These results indicate that the two-cell stage nuclei were unable to transcribe the eIF-1A and Alberich genes when transferred to late one-cell stage cytoplasm. Both of these mRNAs were expressed at very low abundances in the one-cell cytoplasts and nuclear transfer embryos compared with two-cell stage embryos. The failure to detect eIF-1A or Alberich gene transcription in late one-cell nuclear transplant embryos was not likely an artifact (e.g., mRNA degradation during embryo lysis), because abundant maternal G6pd mRNA was detected and the ERV-L and U2afbp-rs mRNAs increased in abundance in these embryos. Thus, the late one-cell stage cytoplasm did not appear to be permissive for transcription of the eIF-1A and Alberich genes.

No significant differences in expression of the maternally derived G6pd mRNA were observed between nuclear transplant embryos and their enucleated cytoplast controls. Expression was greater in one-cell stage embryos than in two-cell stage embryos, as was previously observed [24].

Transcription of the U2afbp-rs and ERV-L genes in untreated late one-cell stage nuclear transplant embryos provides further confirmation that the two-cell stage donor nuclei employed in these studies were developmentally programmed to transcribe an array of genes appropriate for the two-cell stage, as previously observed [32]. Clearly, however, different genes respond with different efficiencies to the late one-cell stage cytoplasm. Both U2afbp-rs and ERV-L were transcribed in the nuclear transplant embryos. The magnitude of the increase in expression of these two genes in nuclear transplant embryos appeared to be smaller than that observed for the TRC [32]. Direct comparisons of the RT-PCR results to the results obtained with the TRC, however, are complicated by the TRC expression being analyzed at the level of protein synthesis, by one-cell embryos exhibiting zero detectable synthesis of this protein, and by TRC protein complex being the most highly synthesized protein in mid two-cell stage embryos, accounting for more than 4% of the total 35S-methionine incorporation in mid two-cell stage embryos and, possibly, indicating an unusually rapid and highly efficient recruitment of the TRC mRNA for translation [42]. The expression of the ERV-L mRNA was very low relative to that in normal two-cell stage embryos, in contrast to the results obtained for U2afbp-rs. Surprisingly, the abundance of neither the eIF-1A mRNA nor the Alberich mRNA was increased significantly in the late one-cell stage nuclear transplant embryos; these two genes appeared not to be transcribed. Thus, although the late one-cell stage cytoplasm supports transcription of some genes (e.g., TRC, U2afbp-rs, and ERV-L), it is not permissive for the transcription of other genes (e.g., eIF-1A and Alberich), and even those genes that are transcribed exhibit different degrees of expression.

These differences in expression following nuclear transplantation could reflect differences in transcription or differences in mRNA accumulation. Given the limited degree of endogenous gene transcription observed by in situ incorporation of bromo-uridine triphosphate (Br-UTP) in one-cell embryos [3], an effect at the level of transcription appears to be most likely for these differences. The inability of the eIF-1A or Alberich genes to be transcribed and the inefficient transcription of ERV-L could reflect a failure of {alpha}-amanitin-treated donor embryos to program these genes fully for transcription. Differences in expression may also indicate that the late one-cell stage cytoplasm does not support efficient transcription of all genes, even those that have been developmentally programmed for expression. Either of these two explanations raises interesting possibilities for understanding the mechanisms that regulate early embryonic gene transcription. If the eIF-1A and Alberich genes are not correctly programmed for transcription in the {alpha}-amanitin-treated embryo, then this would indicate that one or more factors required to transcribe these genes must be produced during one of the previous waves of gene transcription. Although such a relationship between early and later phases of transcription has often been suggested, to our knowledge, direct evidence in support of this relationship has not been obtained. A clear test of this possibility will likely require the development of a suitable in vitro transcription system with which to assay the transcriptional competence of these genes in untreated versus {alpha}-amanitin-treated embryonic nuclei. The alternative possibility, that the one-cell stage cytoplasm is not permissive for transcription of the eIF-1A and Alberich genes, might be explicable by the presence of a transcriptional repressor in the late one-cell embryo that specifically blocks transcription of these genes [43] or by a paucity of factors needed to transcribe these genes. The inefficient transcription of ERV-L may, likewise, reflect limited availability of transcriptional activators for this gene. Our previous studies revealed a need for protein synthesis to activate transcription of housekeeping genes [30], which is consistent with the possibility of a limited availability of positive-acting transcription factors. Other studies have also documented the stage-specific appearance of certain transcription factor activities [41, 44–47]. Further studies employing nuclear transplantation should provide the means for testing these possibilities. Also worth noting is that a restriction in the array of genes that can be activated during the late one-cell stage likely reflects an important regulatory mechanism that helps to ensure that the correct array of genes is activated at this early stage.

Numerous studies have revealed that mammalian genome activation is a multistep process [8, 17, 21, 32, 48]. Our finding that expression of the U2afbp-rs and ERV-L mRNAs in nuclear transplant embryos is sensitive to cycloheximide treatment indicates that protein synthesis facilitates transition of the one-cell stage cytoplasm from an early, transcriptionally nonpermissive state to a permissive state as an early step in the pathway leading to genome activation. This role for protein synthesis in acquisition of the transcriptionally permissive state is reminiscent of a similar role for protein synthesis previously observed for the major genome activation event that occurs during the two-cell stage [30]. The results presented here thus indicate that protein synthesis contributes to genome activation at two different steps in the process and, therefore, is likely to play an important role in the overall control of genome activation.

Protein synthesis may be required indirectly for allowing normal progression through the cell cycle, which in turn may be responsible for regulating the transition. Cycloheximide might also exert indirect effects through other mechanisms related to synthesis of protein phosphorylases or phosphatases, which in turn could affect the activities of transcription factors in the nuclei. One such possibility is through an effect on the phosphorylation state of RNA polymerase II. The {alpha}-amanitin-treated donor nuclei must obtain RNA polymerase II from the recipient cytoplasm for gene transcription to occur. In mouse embryos, the IIO form of the RNA polymerase II subunit RPB1, which has a hyperphosphorylated carboxy terminal domain (CTD), but not the IIA form (i.e., hypophosphorylated CTD), is present during the early one-cell stage, and both forms exist in the late one-cell stage embryo [48]. Cycloheximide treatment reduces expression of the IIO form, but not the IIA form, at the late one-cell stage [48]. Thus, protein synthesis may be required for the expression of an essential protein kinase to provide for expression of the IIO form. Cycloheximide treatment does not appear to have a major effect on either the amount of RPB1 IIA present or on the normal translocation of RPB1 to the nucleus at the two-cell stage [48]. Thus, an indirect effect of cycloheximide on RNA polymerase activity via synthesis of an essential protein kinase is a possibility. Our results indicate, therefore, that protein synthesis to maintain expression of the hyperphosphorylated form of RPB1 may be required for establishing the transcriptionally permissive state in the late one-cell embryo.

Protein synthesis may also be required to permit either stage-specific translational recruitment of maternal mRNAs encoding specific transcription factors or continuous synthesis of other essential transcription factors [30]. We have recently demonstrated both continuous and stage-specific translation of maternal mRNAs encoding such factors as Sp1 and mTEAD-2, respectively [41].

Our previous study indicated that protein kinase A might be involved in the transition to the permissive state [32]. It is conceivable that protein kinase A might affect the transition via a protein synthesis-dependent mechanism by modulating the translational recruitment of maternal mRNAs encoding RNA polymerase II, other transcription factors, or protein kinases. The translational recruitment of maternal mRNAs is under the control of specific mRNA-binding proteins from which the mRNA must be released [49–52]. The binding of these mRNAs may be regulated by protein kinases within the cell, which in turn may be controlled by cell-cycle regulatory proteins [52–55]. Protein kinase A may thus provide a link through which cell-cycle progression controls the timing of the first wave of transcriptional activity by determining the time of appearance or increase in abundance of certain essential transcription factors. Whatever the reason for the requirement for protein synthesis, it appears that the simplest hypothesis, involving a direct effect of protein kinase A on maternal transcription factors, cannot account for the stage-dependent transition to the transcriptionally permissive state, and that the array of maternal proteins present within the early one-cell stage cytoplasm is not adequate to support fully early embryonic gene transcription.

ACKNOWLEDGMENTS

We thank Prof. R.M. Schultz and Dr. Warren Davis for generously providing the cDNA clone for eIF-1A. We also thank Prof. Barbara Knowles and Prof. Davor Solter for their comments on the manuscript.

FOOTNOTES

First decision: 7 March 2001.

1 Supported in part by Public Health Service grants (GM-56682, HD37102, 5 T32 CA09214-20), by a PhD Fellowship from the Max-Planck Society (to M.S.), and by a grant from The Lalor Foundation (to W.N.dV.). Back

2 Correspondence: Keith E. Latham, Department of Biochemistry, 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: April 19, 2001.

Received: February 8, 2001.

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