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BOR - Papers in Press, published online ahead of print January 14, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.023796
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BIOLOGY OF REPRODUCTION 70, 1419–1427 (2004)
DOI: 10.1095/biolreprod.103.023796
© 2004 by the Society for the Study of Reproduction, Inc.

Embryo

Expression of Genes Encoding Chromatin Regulatory Factors in Developing Rhesus Monkey Oocytes and Preimplantation Stage Embryos: Possible Roles in Genome Activation1

Ping Zheng3, Bela Patel3, Malgorzata McMenamin3, Ann Marie Paprocki5, R. Dee Schramm5, Norman G. Nagl, Jr3, Deborah Wilsker3, Xiaomei Wang3, Elizabeth Moran3,4, and Keith E. Latham2,3,4

The Fels Institute for Cancer Research and Molecular Biology3 the Department of Biochemistry,4Temple University School of Medicine, Philadelphia, Pennsylvania 19140 The Wisconsin National Primate Research Center,5 University of Wisconsin-Madison, Madison, Wisconsin 53715


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
One of the most critical events of preimplantation development is the successful activation of gene transcription. Both the timing and the array of genes activated must be controlled. The ability to regulate gene transcription appears to be reduced just prior to the time of the major genome activation event, and changes in chromatin structure appear essential for establishing this ability. Major molecules that modulate chromatin structure are the linker and core histones, enzymes that modify histones, and a wide variety of other factors that associate with DNA and mediate either repressive or activating changes. Among the latter are chromatin accessibility complexes, SWI/SNF complexes, and the YY1 protein and its associated factors. Detailed information about the expression and regulation of these factors in preimplantation stage embryos has not been published for any species. In order to ascertain which of these factors may participate in chromatin remodeling, genome activation, and DNA replication during early primate embryogenesis, we determined the temporal expression patterns of mRNA encoding these factors. Our data identify the predominant members of these different functional classes of factors expressed in oocytes and embryos, and reveal patterns of expression distinct from those patterns seen in somatic cells. Among each of four classes of mRNAs examined, some mRNAs were expressed predominantly in the oocyte, with these largely giving way to others expressed stage specifically in the embryo. This transition may be part of a global mechanism underlying the transition from maternal to embryonic control of development, wherein the oocyte program is silenced and an embryonic pattern of gene expression becomes established. Possible roles for these mRNAs in chromatin remodeling, genome activation, DNA replication, cell lineage determination, and nuclear reprogramming are discussed.

embryo, gene regulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Perhaps the most crucial, and yet least understood, events in the early embryo are those related to transcriptional activation of the embryonic genome. Both the timing and the array of genes activated in the early embryo need to be regulated correctly to ensure proper execution of the developmental program [1–3]. Precocious activation could be associated with abnormal epigenetic modifications [3], while a delay in transcriptional activation could lead to developmental arrest.

Numerous studies in the mouse and other species have documented changes in chromatin structure, and specifically changes in histone content and posttranslational modification [4–7], which are accompanied by overall changes in transcriptional permissiveness [8–13]. Other notable observations include changes in promoter preference and differences in transcription between maternal and paternal pronuclei [5, 14, 15]. The current view is that the balance between histone deacetylation and acetylation becomes shifted in favor of greater histone acetylation around the time of genome activation [2]. Along with changes in histone acetylation, transitions in linker histone content [16– 18], changes in histone methylation [19], and global DNA demethylation [19–21] could also participate in genome activation. Paradoxically, the transcription of exogenous target genes appears to be quite precocious during the period preceding genome activation, with transcriptional enhancers dispensable for this process [22–26], indicating that, along with transcriptional activating events, come other changes that impose a transcriptionally repressive chromatin environment. This paradox is explicable by a model wherein the establishment of a repressive chromatin environment that permits gene-specific regulation is necessary to allow transcriptionally activating changes to be targeted to the correct array of genes [2].

The molecular mechanisms that underlie such changes and that provide correct temporal coordination between transcriptional activation and other key events such as DNA replication have been only partly illuminated. Some studies revealed that progression through S phase is required for major changes in transcriptional activity in the mouse embryo [15, 22, 27]. Other studies have revealed that protein synthesis is required for transcriptional activation [28] and that recruitment of essential maternal mRNAs likely accounts for this requirement [29]. Because the recruitment of masked maternal mRNAs can be regulated by cell cycle regulators [30, 31], temporally regulated recruitment of maternal mRNAs could provide a mechanism for coordinating acquisition of transcriptional capacity with other events, such as cell cycle progression [1, 2]. Products encoded by the recruited maternal mRNAs may then in turn direct the transcription and production of other important regulatory genes as development progresses. Thus, temporal patterns of synthesis of key transcription factors throughout the preimplantation period may regulate the timing of important events during early development that prepare the embryo for implantation [29].

The identities of those transcription factors that participate in the early chromatin remodeling process and in the subsequent transcriptional activation events are largely unknown. In order to understand how the developmental program is executed, it is essential to determine the temporal patterns of expression of genes encoding key transcription factors and to determine which of these genes are expressed maternally, which are expressed at later stages and which display the appropriate stage-specificity of expression to participate in the above processes. Additionally, it is important to understand to what degree such regulatory mechanisms are conserved among different species, and in particular how such mechanisms may contribute to normal development in human and nonhuman primate embryos. Many of the relevant observations to date have been made in species such as the mouse, rabbit, and cow, but few data are available for the human embryo, and a clear paucity of data exist for nonhuman primates. Because primate embryos may differ in fundamental ways from embryos of other species [32, 33], it will be important to acquire data for primate species and to relate those data to data obtained in other species. Knowledge of the factors that exist in healthy primate oocytes and embryos could provide new criteria by which to evaluate oocyte and embryo quality, and thus facilitate improvements in embryo culture and fertility treatments. Last, an understanding of those factors that regulate genome function in fertilized oocytes would provide an understanding of how the oocyte can direct nuclear reprogramming during cloning procedures and how cloning might be made more efficient.

To understand better the molecular controls that regulate preimplantation development during primate embryogenesis, we have developed a new resource, the Non-Human Primate Embryo Gene Expression Resource (PREGER), to permit detailed quantitative gene expression studies in a nonhuman primate species, the rhesus monkey (see accompanying paper [33]). We have used this novel resource to examine the expression of mRNAs encoding a variety of transcription factors in rhesus monkey oocytes and preimplantation stage embryos. Specifically, we have examined the temporal coordination between the expression of these mRNAs and key events such as transcriptional activation and cellular differentiation of the trophectoderm, with particular emphasis on mRNAs encoding transcription factors that play a role in modulating chromatin structure. These include histone acetyltransferases, histone deacetylases, components of the chromatin accessibility complex, members of SWI/SNF transcription regulatory complexes, and other factors that recruit modifiers of chromatin structure, such as YY1. The expression data reveal that the mRNAs encoding certain of these factors are expressed at the appropriate times to play key roles in regulating processes such as genome activation, nuclear reprogramming, DNA replication and repair, and lineage determination.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Oocytes and Embryos

The Wisconsin National Primate Research Center is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, and animal protocols and experiments were approved by the Graduate School Animal Care and Use Committee. The animals were maintained according to recommendations of the Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act with its subsequent amendments.

The isolation and culture of rhesus monkey oocytes and embryos is described in detail in the accompanying paper [33]. Briefly, over 160 samples of oocytes and embryos of various stages and produced by various protocols were obtained. From among this collection, we employed samples of germinal vesicle stage oocytes, in vivo-matured metaphase II oocytes, and in vitro fertilization (IVF)-derived embryos cultured in vitro in HECM9 sequential media [34]. Between 3 and 13 samples of 1–4 oocytes/ embryos were obtained for each stage. It should be noted that, because the entire mRNA population is uniformly amplified during the PCR procedure, the amount of input mRNA (i.e., the range of 1–4 embryos) does not affect the quantitative representation of sequences within the amplified material. As noted in the accompanying paper [33], the embryos collected for inclusion in the PREGER sample set were of high quality and healthy in appearance, with blastomeres displaying uniform granularity. Fragmented embryos were avoided. A minimum of three females were employed to obtain samples for each stage, with the exception of the two- cell stage, for which two females were employed.

cDNA Probes and Hybridization

The cDNA probes employed in these studies are described in Table 1. The cDNA probes were obtained by reverse transcription-polymerase chain reaction (RT-PCR) or from other sources as indicated. The identities of amplified cDNAs were confirmed either by using diagnostic restriction digests or DNA sequencing. Blot preparation, probe preparation, hybridization, and quantitative analyses were performed as described in the accompanying paper [33] and elsewhere [35, 36]. Data were expressed as the mean (±SEM) cpm bound value for each stage/condition of oocytes and embryos included in the analysis. The significance of differences between stages and conditions was evaluated using a t-test.


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  		TABLE 1. Primers and probes used in the study to examine expression of the four gene classes


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of mRNAs Encoding Regulators of Histone Acetylation

Histone acetylation status is a key regulator of chromatin structure and gene transcription. Numerous changes in histone acetylation have been observed in the early embryo and correlated with transcriptional activity, but the identities of the factors responsible have not been examined in any species. To identify those factors present in oocytes and preimplantation stage rhesus monkey embryos, we examined the temporal expression patterns of mRNAs encoding five histone acetyltransferases and five histone deacetylases (Fig. 1). Among the histone acetyltransferases, striking stage-specific differences in expression were observed. The PCAF mRNA displayed two discrete periods of abundant expression, the first being at the eight-cell stage, and the second corresponding to the hatched blastocyst stage. Expression of PCAF mRNA at the eight-cell stage was variable, being elevated in only 5 of 13 samples and thus did not differ significantly from the {alpha}-amanitin-treated embryos. This may reflect the transient nature of its expression at this stage. The GCN5 mRNA was expressed first as a maternal mRNA of somewhat low abundance, judging from the strength of the hybridization signal. GCN5 mRNA expression remained at a low level, and this expression was {alpha}-amanitin insensitive through the eight-cell stage, indicating persistence of the maternal supply. The HAT1 mRNA expression was significantly increased in early blastocysts relative to the small amount of maternal mRNA remaining in {alpha}-amanitin-treated embryos (P < 0.01). The expression patterns observed for HAT1 and GCN5 are quite similar to those reported previously for bovine embryos [37], although our data reveal induction of mRNA at the morula stage, a stage not analyzed in the bovine study. The CBP mRNA was expressed abundantly as a maternal mRNA in oocytes that persisted at least through the eight-cell stage. Based on the strength of the hybridization signals, it appeared that CBP was the predominant member of the class of mRNAs encoding proteins with histone acetyltransferase activity in the oocyte and early embryo. The CBP mRNA declined in abundance between oocyte maturation and the eight-cell stage, and then increased again in abundance with development to the blastocyst stage (P < 0.01). A very faint hybridization signal was obtained using a probe for p300, and this also displayed maternal expression early. The abundance of the p300 mRNA decreased during oocyte maturation (P < 0.001).



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  		FIG. 1. Temporal expression patterns of mRNAs encoding regulators of histone acetylation. Graphs show the relative levels of expression for GV and MII stage oocytes and pronucleate through hatched blastocyst stage embryos produced by in vitro fertilization of oocytes from hCG-stimulated females and then cultured in vitro in HECM9. GV, Germinal vesicle stage oocyte; MII, MII-stage oocyte; PN, pronucleate one-cell stage embryo; 2C, two-cell stage; 8C, eight-cell stage; 8–16C {alpha}Am, 8- to 16-cell stage cultured in {alpha}-amanitin; EB, early blastocyst; XB, expanded blastocyst; HB, hatched blastocyst. Data are expressed as the mean CPM bound ± the SEM. Statistically significant differences in gene expression corresponding to some of the major increases or decreases in expression are denoted by the brackets (for comparisons between stages at the ends of the brackets). Letters a through e indicate P < 0.05, 0.02, 0.01, 0.001, and 0.0001, respectively

Among the histone deacetylases, the predominant mRNAs expressed were those encoding HDAC1 and HDAC2. The HDAC2 mRNA was expressed abundantly in the geminal vesicle (GV) stage oocyte, and diminished in abundance upon maturation (P < 0.01). Expression was sensitive to {alpha}-amanitin at the eight-cell stage. The HDAC2 mRNA appeared to be the most abundantly expressed member of the HDAC family. The HDAC1 mRNA was expressed at near background levels in oocytes and pronucleate through two-cell stage embryos and was then variably induced at the eight-cell stage, with only five samples displaying elevated rates of synthesis compared with the {alpha}- amanitin-treated embryos. Thus, the mean expression levels were not significantly different between treated and untreated embryos at the eight-cell stage (P = 0.19). Expression was significantly greater at the blastocyst stage compared with the treated embryos, indicating transcription of the HDAC1 gene after the eight-cell stage. The predominance of HDAC1 and HDAC2, and the temporal patterns shown here are similar to those reported for bovine embryos, although an apparent species difference exists in that the HDAC1 mRNA was more prevalent than HDAC2 mRNA in the bovine embryo [37]. The HDAC3, HDAC4, and HDAC6 mRNAs were expressed at low levels. HDAC3 mRNA expression displayed no statistically significant changes in expression. The HDAC4 mRNA was poorly represented in oocytes and during early cleavage stages, and a slight but significant increase occurred comparing blastocysts with the {alpha}-amanitin-treated embryos, indicating a low level of transcription initiating by the blastocyst stage. The HDAC4 mRNA expression pattern thus resembled the pattern seen for HDAC1. The HDAC6 mRNA was expressed most prevalently in the oocyte and early cleavage stage embryos, being downregulated in abundance by the eight-cell stage (P < 2 x 10–7).

Expression of mRNAs Encoding Regulators of Chromatin Accessibility

The chromatin accessibility complexes (CHRAC) facilitate the entry of transcription factors into chromatin and also are important for allowing DNA replication [38, 39]. The expression of these complexes, therefore, could be important for both gene transcription and DNA replication during cleavage. We examined the expression of mRNAs encoding five components of the CHRAC (Fig. 2). The ACF1 protein (also known as BAZ1A) appears critical for proper ATP-dependent modification of chromatin structure [38–40]. The mRNA encoding ACF1 was expressed abundantly in oocytes. Its apparent abundance declined (P < 0.02) and then increased again at the eight-cell stage (P < 0.05) in an {alpha}-amanitin-independent manner, possibly indicating regulation at the level of polyadenylation. The ACF1 mRNA then declined significantly in abundance after the eight-cell stage (P < 0.01). The ACF1 protein interacts with CHRAC1 and CHRAC17 to form functional complexes that participate in chromatin remodeling [38–41]. The CHRAC1 mRNA was expressed at a low level in oocytes and early-stage embryos and then at an increased level from the eight-cell stage onward (P < 0.001). Expression at the eight-cell stage was {alpha}-amanitin insensitive, as seen for ACF1, indicating possible coordinate recruitment of these mRNAs. The CHRAC17 mRNA was expressed abundantly in the GV stage oocyte, diminished in abundance during maturation and fertilization (P < 0.0001), and then increased in apparent expression between the pronucleate and eight-cell stages (P < 0.02). This increase appeared to be {alpha}-amanitin sensitive, but this was variable and not statistically significant (P = 0.0635). Expression at later stages was significantly greater than seen in the {alpha}-amanitin-treated embryos (P < 0.01), indicating transcription. Two other factors that interact with ACF1, CHRAC1 and CHRAC17 are the ISWI homologues, SMARCA5 (SNF2H) and SMARCA1 (SNF2L1) [40]. These proteins possess ATPase activity and participate in regulating chromatin access. The SMARCA5 mRNA was expressed predominantly as a maternal mRNA, which diminished in abundance during oocyte maturation (P < 0.05) and then largely disappeared by the eight-cell stage (P < 0.02). Hybridization for the SMARCA1 mRNA was near background throughout development (only one sample of eight-cell embryos produced a significant hybridization signal).



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  		FIG. 2. Temporal expression patterns of mRNAs encoding components of the chromatin accessibility complex. Data and abbreviations are as described in Figure 1

Expression of mRNAs Encoding SWI/SNF-Related Transcriptional Regulators

In addition to the ISWI-related class of ATPases (SMARCA1 and SMARCA5), there exist ATPases of the SWI2/SNF2 class. These ATPases, together with their associated factors (i.e., the SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin class of transcription factors) encompass a diverse group of proteins with homology to the components of the Drosophila brahma complex [42, 43]. The complexes formed by these proteins mediate either activating or repressing changes in gene transcription, through alterations in chromatin structure mediated by ATP-dependent movement of nucleosomes or DNA bending and looping [44–54]. To learn which of these proteins might participate in genome activation and subsequent gene regulation in the early embryo, we analyzed their patterns of expression (Fig. 3). Functionally and structurally related members of the family displayed opposing and complementary patterns of expression. For example, of the mRNAs encoding SMARCA2 and SMARCA4 (also known as BRM and BRG1), the two alternative ATPases of SWI/SNF complexes, SMARCA2 mRNA was expressed exclusively as a maternal transcript until the hatched blastocyst stage (P < 0.05), whereas SMARCA4 was transcriptionally induced by the morula stage (P < 0.01). Both mRNAs displayed significant declines in apparent abundance during oocyte maturation (P < 0.0001). The FANCA mRNA, which encodes a BRG-1 interacting protein possibly involved in recruiting SWI/SNF complexes to DNA [55], was expressed at a low level, with {alpha}-amanitin-sensitive gene transcription being evident at the eight-cell stage (P < 0.05) and beyond (P < 0.02). Complementarity in expression was also seen for the mRNAs encoding p270 (SMARCF1/ARID1A) and ARID1B (originally named KIAA1235 [56]), two closely related DNA binding proteins of the ARID class that are alternative components of the SWI/SNF complexes and that are typically coexpressed in somatic cells (X. Wang et al., unpublished data). SMARCC1 (BAF155) mRNA was expressed in GV stage oocytes, was then nearly eliminated (P < 0.0001), and was later induced at the hatched blastocyst stage (P < 0.01) when ARID1B was also induced. Another contributor to SWI/SNF complex function, SMARCE1 (BAF57), was down-regulated during oocyte maturation (P < 0.0001)) and then up-regulated at the morula stage (P < 0.0001). In contrast, BR140, a bromo-domain protein, was expressed predominantly as a maternal mRNA, down-regulated between the GV and eight-cell stages (P < 1 x 10–6), but with a low level of transcription evident at the eight-cell stage (P < 0.05). The SMARCD1 (BAF60A) mRNA displayed increased expression at the two-cell stage (P < 0.001), but otherwise was barely detectable. Thus, among this group of genes, some were expressed predominantly as maternal mRNAs, while others were expressed primarily as embryonic transcripts, with functionally related members often showing opposing patterns of expression.



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  		FIG. 3. Temporal expression patterns of mRNAs encoding SWI/SNF-related transcription regulators and associated factors. Data and abbreviations are as described in Figure 1

Expression of mRNAs Encoding YY1 and Associated Factors

The YY1 transcription factor is ubiquitously expressed and exerts either activating or repressing effects on transcription by associating with YAF2 or RYBP, respectively [57, 58]. YY1 association with proteins such as YAF2 and RYBP can lead to the recruitment of histone acetylases, histone deacetylases, and histone methylases to DNA to alter chromatin structure [59, 60]. Thus, YY1 and its partners constitute another important group of proteins that can affect chromatin structure. The YY1 mRNA was expressed throughout development (Fig. 4). By the morula stage, transcription was {alpha}-amanitin sensitive, indicating active expression from the embryonic genome. The RYBP mRNA was expressed at a low level until the hatched blastocyst stage, when it was upregulated (P < 0.02). Expression of RYBP mRNA at the eight-cell stage was {alpha}-amanitin sensitive (P < 0.05). The YAF2 mRNA, by contrast, was expressed as a maternal mRNA that declined in abundance with development to the early blastocyst stage (P < 0.01). Thus, the YAF2 and RYBP mRNAs display complementary patterns of expression.



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  		FIG. 4. Temporal patterns of expression of YY1 and associated factors. Data and abbreviations are as described in Figure 1


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The data presented here reveal for the first time in any primate species the predominant members of four key classes of chromatin regulatory transcription factors. With the exception of the HDACs, which were examined in bovine embryos [37, 61], this study is the first to examine the expression of these classes of transcription factors in any species of mammalian embryo. Our observations thus provide novel information about which factors are expressed at the appropriate stages to play key roles in processes such as genome reprogramming, genome activation, DNA replication, DNA repair, and cell lineage commitment in the early preimplantation embryo, and what regulatory activities are likely lacking during this period. Because the data presented are for a nonhuman primate species, they provide significant new insight into how early embryogenesis is likely regulated in the human.

One of the most striking trends apparent in the data presented here is that, although the transcription factors analyzed are widely expressed and often considered to be constitutively expressed in somatic cells, some of the mRNAs are poorly expressed or undetectable at all of the stages analyzed, and many others show stage-specific expression. Moreover, the stage specificity of expression divides many of the expressed mRNAs into either of two reciprocal categories, displaying predominantly maternal expression or strong induction in the embryo. These patterns of expression appear for each of the four groups of mRNAs analyzed and are consistent with discrete functions in important processes (Table 2). The division of the mRNAs of all four classes into the two temporally distinct groups suggests a possible mechanism for mediating the switch from maternal control to embryonic control of development. Those factors that are expressed in the oocyte may represent a legacy remaining from the oocyte differentiation and development pathway, but may also mediate early events that could support genome activation. Conversely, those factors that are upregulated during development are likely to play critical roles in establishing an embryonic pattern of gene regulation.


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  		TABLE 2. Distribution of some analyzed mRNAs between the predominantly maternal class and the embryonically induced class of message

Each of the four classes of mRNAs examined display members that are induced at the time of embryonic genome activation [62] and at the blastocyst stage. Induction of a number of these mRNAs at the eight-cell stage suggests a possible role in promoting or propagating the major embryonic genome activation event, which occurs between the six-cell and eight-cell stages. Interestingly, the ACF1 and CHRAC1 mRNAs display {alpha}-amanitin-insensitive increases in apparent abundance, consistent with possible polyadenylation and recruitment at that stage [36]. Such recruitment could provide for a stage-specific increase in the availability of these proteins, thereby facilitating opening of chromatin for transcription. The induction of the CHRAC17 gene by the eight-cell stage could provide further capacity for CHRAC function, thereby facilitating both gene transcription and continued DNA replication and cell cycle progression. The expression of CHRAC complexes in the oocyte could also contribute to the oocytes' ability to reprogram nuclei after somatic cell nuclear transfer.

Our results also point to specific members of the SWI/ SNF family of transcriptional regulators as possible key players in genome activation. The induction of the SMARCA4 (BRG1) ATPase-bearing protein at the morula stage and the induction of the SMARCE1 (BAF57) accessory protein may be especially relevant for further transcriptional activation of many genes, by permitting ATP-dependent chromatin remodeling. These proteins may also contribute to nuclear remodeling during cloning. Interestingly, homozygous SMARCA4 knockout mouse embryos arrest during preimplantation development [63], consistent with an essential function during cleavage. The up-regulation of SMARCA4 contrasts with the loss of the maternal SMARCA2 mRNA encoding the alternative ATPase for the SWI/ SNF complex. Homozygous deficiency for SMARCA2 in mice, however, is not lethal, but homozygous mutants display growth defects, possibly related to altered regulation of the cell cycle [64]. Thus, SMARCA4 may play a predominant role during preimplantation development, whereas SMARCA2 may be important as a maternal factor in the oocyte and early embryo for regulating the cell cycle. Because SMARCA2 may also regulate association of cohesin with chromatin [49], its expression as a maternal factor in the oocyte may be important for regulating chromosome pairing. It was suggested that SMARCA4 expression may compensate for an absence of embryonic SMARCA2 expression [64]. To date, however, it is not known whether a maternal effect phenotype exists for BRM homozygous null females. It is also worth noting that the transitions from SMARCA2 predominance in the oocyte and early embryo, to SMARCA4 predominance after fertilization and through the early blastocyst stage, and finally to coexpression of these two mRNAs in hatched blastocysts may constitute key steps, first in the transition from maternal to embryonic control of development and second during the differentiation of the blastocyst cell lineages. This is because complexes formed with SMARCA4 may activate a different array of genes than that activated by SMARCA2 in the embryo, as they do in other cells [48].

The induction of HAT1 and PCAF at the eight-cell stage, though variable, may also serve to promote gene transcription by increasing the available supply of histone acetylating activities. This increase appears to be balanced by an increase in HDAC1 and HDAC2 mRNA expression. This balance may be needed in order to limit transcriptional activation to the correct array of genes.

The upregulation of p270, ARID1B, SMARCA2, SMARCC1, PCAF, HDAC1, and RYBP by the blastocyst stage likely establishes within the embryonic cells at that stage an array of expression of mRNAs more typical of somatic cells and may be related to cell lineage commitment within the developing blastocyst. In this context, it is interesting that SMARCC1 (BAF155) deficiency in mice is associated with preimplantation lethality and a defect in the formation of the inner cell mass lineage [65]. Additionally, the up-regulation of SMARCE1 (BAF57) at the morula stage could also contribute to cell lineage specification, as this protein participates in lineage bifurcation and gene switching in other cell types [44, 66]. The induction of SMARCA2 at the blastocyst stage may also contribute to lineage-specific changes in gene expression. Interestingly, the noticeably low level of expression of SMARCA5 and SMARCA1 indicates that the widely expressed ISWI components, which play important roles in promoting cellular proliferation and differentiation, respectively, may not play a prominent role during blastocyst formation and may only become key factors during postimplantation life.

Other mRNAs, such as YY1, CBP, and HDAC3, do not readily fit into either maternal or embryonic transcript category. Their constitutive presence as members of both categories may reflect roles for the corresponding proteins during both periods of development. The abundant expression of YY1 in the oocyte may signify a function in promoting gene transcription, but could also signify other functions, such as contributions to DNA repair during the postfertilization period [67, 68]. HDAC3 interacts with a variety of proteins, with which it represses transcription [e.g., 69, 70], including RB, with which it operates to control cell proliferation via PPAR-gamma [71], a function that could also contribute to cell cycle control and DNA repair.

The data presented here clearly reveal certain members of each class of transcription factors as prominent during either the maternally controlled or the embryonically controlled portions of preimplantation development. This is the first detailed study addressing in a common set of samples the expression patterns of each of these classes of factors. As such, these observations provide the necessary foundation for designing further studies to understand the specific roles played by these factors in the early embryo. Of particular interest will be the examination of the functions of maternally expressed mRNAs on processes such as nuclear reprogramming and initial genome activation, and the effects of early embryonically expressed transcripts on continued genome expression and regulation. Alterations in the expression of some of these mRNAs in oocytes of different developmental potentials (unpublished) suggest that the expression of such mRNAs could provide useful markers of oocyte quality. The data presented here provide the basis for undertaking key functional studies in oocytes and embryos of both human and nonhuman primate species.


   FOOTNOTES
 
1 Supported by a grant from the NIH/NCRR (RR15253) to K.E.L. Back

2 Correspondence: Keith E. Latham, 3307 North Broad Street, Philadelphia, PA 19140. FAX: 215 707 1454; klatham{at}temple.edu Back

Received: 30 September 2003.

First decision: 3 November 2003.

Accepted: 5 January 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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