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BOR - Papers in Press, published online ahead of print February 11, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.022970
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BIOLOGY OF REPRODUCTION 70, 1701–1709 (2004)
DOI: 10.1095/biolreprod.103.022970
© 2004 by the Society for the Study of Reproduction, Inc.

Embryo

Transcription Factor Expression Patterns in Bovine In Vitro-Derived Embryos Priorto Maternal-Zygotic Transition1

Christian Vigneault, Serge McGraw, Lyne Massicotte, and Marc-André Sirard2

Centre de Recherche en Biologie de la Reproduction, Département des Sciences Animales, Université Laval,Québec, Canada G1K 7P4


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Maternal-zygotic transition (MZT) is a complex phenomenon characterized by the initiation of transcription in the embryo and the replacement of maternal mRNA with embryonic mRNA. In order for this to occur, transcriptional activation requires various factors and conditions. Our hypothesis is that the lack of transcription in the bovine pre-MZT embryo is due, in part, to an incomplete or dormant transcriptional apparatus. Therefore, in accordance with this hypothesis, functioning transcriptional mechanisms should appear in the eight-cell bovine embryo to facilitate embryonic transcription during the MZT. With this in mind, we examined the presence of selected transcription factors during preimplantation embryo development to establish how their transcript levels change in bovine pre-MZT embryos. To achieve this goal, real-time reverse transcription-polymerase chain reaction was used to quantify the mRNA level of several different transcription factors (YY1, HMGA1, RY-1, P300, CREB, YAP65, HMGN1, HMGB1, NFAR, OCT-4, TEAD2, ATF-1, HMGN2, MSY2, and TBP) in germinal vesicle (GV) and metaphase II (MII) bovine oocytes and in two-, four-, eight-cell, and blastocyst stage embryos produced in vitro. Our results demonstrate that all genes examined can be grouped into five different categories according to their mRNA expression patterns at the developmental stages observed. To summarize, all transcription factors studied were present in pre-MZT embryos and the expression pattern of many of them suggest a potential role in MZT.

early development, embryo, gene regulation, in vitro fertilization, oocyte development


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
As observed in other species, the mRNA and protein content of the bovine oocyte supports the embryo's first instances of life. It is believed that the mRNAs and proteins acquired by the oocyte during its growth and final maturation allow the zygote to go through the early stages of embryo development up until the moment when the embryo produces these factors on its own. The point at which embryonic transcription begins and maternal mRNA is replaced by embryonic mRNA is referred to as the maternal- zygotic transition (MZT). This takes place at different periods, depending on the species (reviewed in [1, 2]). For example, MZT occurs from the end of the one-cell stage until the two-cell stage in mice [3], at the four- to eight- cell stage in humans [2], and in 4000- to 8000-cell embryos (stage 8–8.5) in Xenopus [4]. Although the bovine MZT occurs at the 8- to 16-cell stage and is characterized by a major onset of transcription, minor transcription is observed as early as the one-cell embryo [5]. The MZT is thought to be an important and limiting step of development because it coincides with a developmental block observed in embryos cultured in vitro [6].

Many components and conditions are likely required to enhance transcription in the young embryo and most still remain to be identified. Evidence in mice suggest that DNA replication during embryo cleavage is a triggering event for MZT [7]. However, in the bovine, MZT occurs after three embryonic divisions, suggesting another level of regulation. Therefore, in species with a MZT occurring after more than one cleavage, pre-MZT embryos' transcriptional silence is probably due to a transcriptional nonpermissive chromatin state and inefficient transcriptional apparatus.

Different factors are involved in chromatin remodeling to produce a transcriptional permissive chromatin state. Histone acetyltransferases (HAT) are proteins that acetylate the histone tails of the nucleosome and consequently relax the chromatin, leading to DNA that is more accessible to transcription factors (reviewed in [8]). Some studies have already revealed the importance of histone acetylation in MZT [9, 10] and expression studies of HAT throughout bovine embryo development have been carried out [11]. In addition to HAT, other factors may promote transcription by modifying chromatin conformation. This is the role of high mobility group (HMG) proteins, a family of chromosomal proteins that act as a structural element of the chromatin without being histones. HMG activity tends to produce a chromatin conformation that improves transcription [12]. In general, a balance between many chromatin-remodeling factors is probably necessary for transcription.

Once the chromatin conformation is adequate and permits the binding of transcription factors, these factors must be present and active to bind this DNA and transcribe the required genes. Transcription factors may be separated in two basic categories: general transcription factors and specific factors. General transcription factors encompass those required for basal transcription by the RNA polymerase II and include the TATA-binding protein (TBP) (reviewed in [13]). The other transcription factors and coactivators are proteins that bind specific DNA sequences in the promoters or enhancers of genes, or bind other transcription factors to recruit the basal transcription machinery to the promoter and facilitate transcription [13, 14].

It can be hypothesized that a virtual lack of transcription in the bovine embryo, before the eight-cell stage, is in part due to an absent or inefficient transcriptional apparatus. In the present study, we investigate the mRNA expression patterns of 15 different transcription factors believed to play a potential role in early transcription during bovine embryo development. A real-time RT-PCR (reverse transcriptase- polymerase chain reaction) approach was used to quantify the transcript level of each factor at different bovine preimplantation embryonic stages from germinal vesicle (GV oocytes) to blastocysts. The factors studied (listed in Table 1) are from many different families and were chosen for their suspected role in MZT based on previously published data showing their presence or activity around the MZT in other species.


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  		TABLE 1. Information on the transcription factors studied

The 15 genes of interest evaluated in the present study were allocated to five different groups dependent on the appearance of their respective expression patterns. This study represents the first known attempt to evaluate the presence of transcription factors in pre-MZT embryos and opens the door to future studies on factors with potential roles in bovine MZT based on their mRNA expression patterns. It is also suspected that a combination of different factors is required for such dramatic changes in gene expression, supporting the multigene analysis approach of this study.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Note that all chemicals were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise stated.

Oocytes Recovery and Selection

Bovine ovaries were collected at a commercial slaughterhouse and transported to the laboratory in a 0.9% NaCl aqueous solution containing antimycotic agent. Cumulus-oocyte complexes (COCs) from 3- to 6-mm follicles were collected with an 18-guage needle attached to a 10-ml syringe. Healthy COCs with at least five layers of cumulus were selected to proceed. The cumulus cells were removed mechanically by vortexing. The denuded oocytes were placed in PBS solution and washed generously, at least three times, to ensure the absence of contamination by cumulus cells. Groups of 40 GV oocytes were then frozen in a minimal volume of PBS and stored at –80°C until RNA extraction.

In Vitro Maturation

COCs with the same characteristics described above for GV oocytes were put through in vitro maturation after three washes in HEPES-buffered Tyrode lactate medium (TLH) supplemented with 0.3% bovine serum albumin (BSA fraction V), 0.2 mM pyruvic acid, and 50 µg/ml gentamicin. Groups of 10 COCs were placed in droplets of media under mineral oil. Each droplet consisted of 50 µl of maturation medium composed of modified synthetic oviductal fluid (SOF) medium [40] with 0.8% BSA, modified Eagle medium (MEM) nonessential amino acids (Gibco BRL, Burlington, ON, Canada), MEM essential amino acids (Gibco), as well as 1 mM glutamine supplemented with 0.5 µg/ml FSH, 5 µg/ml LH, and 1 µg/ ml 17ß-estradiol. The droplets containing COCs were incubated in a humidified atmosphere for 24 h at 38.5°C with 5% CO2. Metaphase II (MII) oocytes were collected at that time and mechanically denuded by vortexing. The oocytes were washed at least three times with PBS to completely remove any contaminating cumulus cells. The MII oocytes were then frozen in groups of 40 in a minimal volume of PBS and stored at –80°C until RNA extraction.

In Vitro Fertilization

For in vitro fertilization, five matured COCs were added to 48-µl droplets under mineral oil. The droplets were composed of modified Tyrode lactate medium supplemented with 0.6% BSA fatty acid free (Sigma-Aldrich), 0.2 mM pyruvic acid, 10 µg/ml heparin, and 50 µg/ml gentamycin. Prior to transfer, the COCs were washed twice in TLH medium. Once transferred, 2 µl of PHE (1 mM hypotaurine, 2 mM penicillamine, 250 mM epinephrine) were added to each droplet ~10 min before semen was added. The semen used consisted of a cryopreserved mixture of ejaculates from three bulls (Centre d'Insémination Artificielle du Québec, St-Hyacinthe, QP, Canada). The semen was thawed in 37°C water for 1 min, put on a discontinuous Percoll gradient (2 ml of 45% Percoll over 2 ml of 90% Percoll) and centrifuged at 700 x g for 30 min at 26°C. The pellet was resuspended in 1 ml of modified Tyrode medium and centrifuged at 250 x g for 5 min at 26°C. The supernatant was discarded and the spermatozoa were resuspended in IVF medium after being counted on a hemocytometer to obtain a final concentration of 1 x 106 cells/ml. Finally, 2 µl of the sperm suspension were added to each droplet and the incubation took place in a humidified atmosphere at 38.5°C in 5% CO2 for 15–18 h.

In Vitro Culture

Following fertilization, presumptives zygotes were mechanically denuded by repeated pipetting, washed three times in PBS containing 0.3% BSA for complete removal of cumulus cells from solution, and transferred to culture droplets (50 µl) in groups of 20–30 embryos. Embryo culture was performed in modified synthetic oviduct fluid (SOF 1) under mineral oil at 38.5°C in 5% CO2 in a reduced oxygen atmosphere (7%) with high humidity. SOF 1 medium was replaced after 72 h by SOF 2 to prevent toxicity due to ammonium accumulation resulting from amino acid degradation. The SOF 1 medium contained 0.8% BSA, MEM nonessential amino acids, 1 mM glutamine, 1.5 mM glucose, and 10 µM EDTA. The SOF 2 medium contained 0.8% BSA, MEM nonessential amino acids, MEM essential amino acids, 1 mM glutamine, and 1.5 mM glucose. The effectiveness of the SOF system for bovine in vitro embryo development has already been shown [41]. The two-, four-, and eight-cell embryos were collected 36, 48, and 72 h postfertilization, respectively, and blastocysts were collected after 7 days of development. All were washed three times in PBS, collected in groups of 40 in small volumes of PBS, frozen, and stored at –80°C until RNA extraction. Note that all collected embryos were derived from populations of embryos that had cleaved at 36 h postfertilization. This criterion was used to identify embryos of superior quality. All oocyte and embryo pools were collected and analyzed in triplicates and the totality of the embryos was collected from seven different fertilizations and at least three fertilizations were done for each stage of development.

RNA Extraction and cDNA Preparation

GFP RNA was transcribed from a partial GFP sequence cloned into pGEM-T easy (Promega, Madison, WI), to which a short poly(A) tail of 21 base pairs (bp) was added. The GFP fragment that we cloned was isolated from the phGFP-S65T vector (Clontech, Palo Alto, CA) and corresponds to the sequence of the fragment between bases 892 and 1598 of the GenBank accession number sequence U43284. This exogenous RNA was produced by in vitro transcription of the construct using the AmpliScribe T7 High Yield Transcription Kit (Epicentre, Madison, WI). One picogram of this exogenous RNA containing a poly(A) tail was added to each pool of oocytes and embryos before RNA extraction. Thus, the RNA extractions of the oocytes or embryo pools containing GFP RNA were performed using the Absolutely RNA Microprep Kit (Stratagene, La Jolla, CA) and the RNA was recovered by two subsequent 50-µl elutions with warmed (60°C) elution buffer provided in the kit. The RNA extraction procedure includes a DNase I treatment to remove genomic DNA. The RNA was precipitated with 10 µl of 3 M sodium acetate pH 5.2, 2.5 µl of 1 mg/ml linear acrylamide (Ambion, Austin, TX), and 100 µl of 100% isopropanol. The pellets were washed with cold EtOH 75%. Air-dried pellets were dissolved in 14.75 µl of 1.36 µM oligodT(18) (to obtain a final concentration of 1 µM after addition of the reverse transcriptase mix). To denature the RNA and remove secondary structures, the RNAs were heated at 65°C for 5 min and then quenched rapidly on ice for 3 min. A mix containing 2 µl of Omniscript 5x Buffer (Qiagen, Mississauga, ON, Canada), 2 µl of 50 µM dNTPs (Qiagen), 0.25 µl of 40U/µl of RNASIN (Promega), and 1 µl of Omniscript Reverse Transcriptase (Qiagen) was then added to each tube. The reactions were then incubated at 37°C for 2 h.

Real-Time Polymerase Chain Reaction

The primers for each gene were designed using Primer3 web interface [42] and from consensus sequences generally derived from human and mouse sequences from NCBI. Primer sequences are shown in Table 2. For each gene examined, a standard curve, consisting of PCR products purified with the QIAquick PCR Purification Kit (Qiagen) and quantified with a spectrophotometer, was included in the run. The standard curve consisted of four standards of the purified PCR products diluted from 0.1 pg to 0.1 fg. Real-time PCR was executed on a Lightcycler apparatus (Roche Diagnostics, Laval, QC, Canada) using SYBR green incorporation. The reaction was performed in capillaries and in a final volume of 20 µl (Roche). Each capillary contained the cDNA corresponding to a single oocyte or embryo and a reaction mixture consisting of 0.5 µl of 10 µM of each primer, 1.6 µl of 25 mM MgCl2 (final concentration of 3 mM), 2 µl of the SYBR green mix containing dNTPs, FastStart DNA polymerase enzyme, and buffer (Roche). The PCR conditions used for all genes were as follows: denaturing cycle of 10 min at 95°C; 40–50 PCR cycles (denaturing: 95°C for 1 sec; annealing: temperature [see Table 2] for 5 sec; extension: 72°C for 20 sec); a melting cycle consisting of 95°C for 1 sec, 70°C for 30 sec, and of a step cycle starting at 70°C up to 95°C with a 0.2°C/ sec transition rate; a final cooling cycle of 40°C for 30 sec. The cDNA quantification was performed using Lightcycler Software Version 3.5 (Roche) with comparison with the standard curve. The first gene to be quantified was the exogenous GFP. The GFP quantity obtained for each pool was used to correct the values obtained for each gene. The pool showing the highest GFP level was designated the reference pool and the values of GFP obtained from each pool were then divided by the reference pool value. The values obtained for each gene in each pool were then divided by the correcting value of the corresponding pool. These calculations compensate for experimental errors caused by the technique or the materials used for the RNA extraction and reverse transcription. The real- time PCR product specificity was confirmed by analysis of the melting curve given by the Lightcycler software (Roche). The products were then electrophoresed on an agarose gel and were sequenced to confirm that the proper product was amplified.


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  		TABLE 2. Information on primers used for RT-PCR experiments

Statistical Analysis

Normalization of the results obtained for individual genes, within each pool of cDNA, was performed by calculating each as a ratio to the level of GFP RNA. Data are presented as mean ± SEM. Statistically significant differences in mRNA level between each developmental stage were calculated by ANOVA and least significant difference test. Differences were considered statistically significant at the 95% confidence level (P < 0.05).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Our results show that all the factors examined are present throughout the period studied (i.e., from GV oocytes to the blastocyst stage). The transcription factors examined were classified into five groups, based on their expression patterns throughout these developmental stages. The results have been presented in five different figures (Figs. 1 through 5) and significant differences between each group can be observed. Figures 1, 2, and 3 show genes with similar expression patterns. Indeed, the mRNA level of these genes decreases from the GV oocyte stage up until the eight-cell stage, where it reaches it lowest level, according to the stages studied. Also, for all these genes, except NFAR, a significant increase is observed in the blastocyst level compared with the eight-cell stage level. The primary difference between the first three groups (Figs. 1 through 3) is the mRNA profile for the GV oocytes to the eight- cell embryos. For the transcription factors HMGA1 and YY1 (Fig. 1), one can see a major decrease of the mRNA level during oocyte maturation. While for RY-1, CREB, P300, HMGN1, and YAP65, the first reduction in mRNA level is observed between MII and the two-cell stage (Fig. 2). Although a major decrease in RY-1 mRNA level occurs after oocyte maturation, a slight decrease during maturation is also observed. Finally, for the genes presented in Figure 3, the maternal mRNA level undergoes it first significant decrease later in development. That is, for HMGB1, NFAR, and TEAD2, this reduction occurs at the four-cell stage, while for OCT-4, it occurs at the eight-cell stage. The group of genes shown in Figures 1 through 3 present expression patterns very similar to those of various housekeeping genes during the same period [43]. These patterns are characterized by a relatively high level of mRNA in the GV oocyte, followed by a decrease until the eight-cell stage, caused by the turnover of the maternal supply. Then finally, an expression of these genes is observed after the MZT, deduced from the high level of mRNA at the blastocyst stage compared with eight-cell stage.



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  		FIG. 1. Quantification by real-time RT-PCR of YY1 and HMGA1 in bovine oocytes and embryos cultured in vitro. Letters below histograms represent the developmental stages studied: G = GV oocytes; M = MII oocytes; 2 = two-cell embryos; 4 = four-cell embryos; 8 = eight-cell embryos; and B = blastocysts. Each developmental stage was done in triplicate and the amounts of mRNA shown represent the quantity of transcript corrected with the GFP value obtained for each pool. Different letters indicate a significant difference (P < 0.05)



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  		FIG. 2. Quantification by real-time RT-PCR of RY-1, P300, CREB, YAP65, and HMGN1 in bovine oocytes and embryos cultured in vitro. Letters below histograms represent the developmental stages studied: G = GV oocytes; M = MII oocytes; 2 = two-cell embryos; 4 = four-cell embryos; 8 = eight-cell embryos; and B = blastocysts. Each developmental stage was done in triplicate and the amounts of mRNA shown represent the quantity of transcript corrected with the GFP value obtained for each pool. Different letters indicate a significant difference (P < 0.05)



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  		FIG. 3. Quantification by real-time RT-PCR of HMGB1, NFAR, OCT-4, and TEAD2 in bovine oocytes and embryos cultured in vitro. Letters below histograms represent the developmental stages studied: G = GV oocytes; M = MII oocytes; 2 = two-cell embryos; 4 = four-cell embryos; 8 = eight-cell embryos; and B = blastocysts. Each developmental stage was done in triplicate and the amounts of mRNA shown represent the quantity of transcript corrected with the GFP value obtained for each pool. Different letters indicate a significant difference (P < 0.05)

In contrast, Figure 4 shows a single transcription factor, ATF-1, which has a constant level of mRNA in all embryonic stages examined. Unlike the other factors studied, ATF-1 shows no significant decrease in mRNA level at any stage. We are unable to determine at this point if ATF-1 transcripts observed at the eight-cell stage are of maternal or embryonic origin.



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  		FIG. 4. Quantification by real-time RT-PCR of ATF-1 in bovine oocytes and embryos cultured in vitro. Letters below histograms represent the developmental stages studied: G = GV oocytes; M = MII oocytes; 2 = two-cell embryos; 4 = four-cell embryos; 8 = eight-cell embryos; and B = blastocysts. Each developmental stage was done in triplicate and the amounts of mRNA shown represent the quantity of transcript corrected with the GFP value obtained for each pool. Different letters indicate a significant difference (P < 0.05)

Finally, the last group illustrated in Figure 5 includes transcription factors HMGN2, TBP, and MSY2 that are present at a higher level in oocytes and two- and four-cell embryos compared with eight-cell embryos and blastocysts. Unlike for the other factors evaluated, the mRNA level of these genes do not increase significantly after embryonic genome activation or at the blastocyst stage. However, there is a significant decrease in the mRNA level during the meiotic maturation, after which the mRNA level remains stable until it decreases at the eight-cell stage and remains low in the blastocyst stage.



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  		FIG. 5. Quantification by real-time RT-PCR of HMGN2, MSY2, and TBP in bovine oocytes and embryos cultured in vitro. Letters below histograms represent the developmental stages studied: G = GV oocytes; M = MII oocytes; 2 = two-cell embryos; 4 = four-cell embryos; 8 = eight-cell embryos; and B = blastocysts. Each developmental stage was done in triplicate and the amounts of mRNA shown represent the quantity of transcript corrected with the GFP value obtained for each pool. Different letters indicate a significant difference (P < 0.05)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
All of the genes evaluated in this study are present in mRNA form in all pre-MZT embryo stages. This suggests that the early embryo could translate these mRNAs into proteins to use these factors during development. Of interest is the fact that the highest level of mRNA is found in the GV oocytes for all the genes examined except for HMGB1, OCT-4, and TEAD2. These three factors tend to be present at their highest level in the blastocyst. This indicates that the oocyte contains large stocks of mRNA of each factor that can be used for translation if the corresponding protein is required for transcription in early embryo development. Furthermore, given that they are strongly present in the oocyte and their presence diminishes up until MZT, one would assume that these factors are of maternal origin. Nevertheless, an important point to consider is that this study was done on in vitro-cultured embryos. It has been shown recently that differences exist between in vivo- and in vitro-cultured oocytes and embryos, especially in gene expression [44, 45]. It is important to consider this while interpreting the results of this study.

In the case of HMGA1 and YY1 (Fig. 1), the mRNA population diminishes during oocyte meiotic maturation. It has been shown that elevated protein synthesis occurs at the GVBD period in bovine oocytes during in vitro maturation [46]. HMGA1 and YY1 mRNA recruitment by the translation machinery and subsequent degradation during maturation could explain the mRNA diminution observed, as this commonly occurs to many transcripts when they are translated [47, 48]. After this initial decrease, the mRNA level is stable from the MII oocyte to the four-cell stage and then declines once again at the eight-cell stage. Assuming that a diminution in mRNA level indicates mRNA translation, this suggests the need for these factors in early steps of development for postmaturation oocytes or early embryos. Thus, it appears that, in bovine embryos, YY1 could be involved in pre-MZT transcription if it is translated during meiotic maturation. Furthermore, if YY1 is present at the eight-cell stage or is translated at the four- or eight-cell stages, it could participate in the MZT. Previous expression studies of HMGA1 in mouse embryos have shown that its mRNA is present before and during the MZT and disappears soon after at the eight-cell stage [49]. In the present study, we do not observe this response in the bovine system, as the level of HMGA1 transcript is relatively low at the MZT. An important point to consider is that our primers amplified the three known isoforms of HMGA1 (HMGA1a, HMGA1b, and HMGA1c). Nevertheless, our results suggest that HMGA1 is present at the stages evaluated and could be involved in chromatin relaxation to promote pre-MZT transcription or even in the chromatin remodeling of the male pronucleus.

The expression patterns of RY-1, P300, CREB, YAP65, and HMGN1 illustrated in Figure 2 are slightly different from patterns presented in Figure 1. In this case, the first significant decrease in mRNA level occurs between the MII oocyte and two-cell embryo stage, suggesting a possible production of proteins for pre-MZT transcription and the following steps of development. The other mRNA decrease happens between the four- and eight-cell stages, which could be related to the translation of new proteins implicated in embryonic gene activation. With its special constitution, it is possible that RY-1 is implicated in the rearrangement of pronucleus chromatin following fertilization or mRNA splicing following transcription in early embryos [19]. The importance of this protein would explain why oocytes with good developmental competence contain higher amounts of RY-1 than poorer ones [18]. Considering that p300 and CREB are master switch genes and that we have shown they are present during early embryo development, one could propose that they play a significant role in transcription before or during the MZT. Although an EST database analysis on two-cell mouse embryos revealed the presence of YAP65 [25], no studies describing the expression patterns of YAP65 during preimplantation embryo development have been published to date. Thus, the YAP65 expression pattern in bovine embryonic development identified in this study is the first to be published. These results show the presence of YAP65 in mammalian early development and imply a role for this factor in the MZT. For the remaining gene, HMGN1, expression studies in the mouse have revealed an elevated transcript level in the GV oocytes and the lowest level in the two-cell embryos [27]. If the MZT timing difference between the mouse and bovine are considered, these results coincide with those of the current study in which the lowest quantity of HMGN1 transcript is found in the eight-cell bovine embryo. Because our results are consistent with those obtained in the mouse, one could infer a role for HMGN1 in bovine embryos transcription, principally the MZT.

The group of factors presented in Figure 3 demonstrate a constant mRNA level from the GV oocytes to the two- cell embryos for HMGB1, NFAR, and TEAD2 or up until the four-cell stage in the case of OCT-4. The fact that this mRNA is conserved for such an extended period (12–36 h considering the moment of collection of the embryos) reinforces the idea that these genes could be involved in the MZT. HMGB1 expression studies in mouse embryos have revealed a very weak presence of HMGB1 mRNA and protein in one- and two-cell embryos compared with other stages [50]. These results suggest that HMGB1 is probably not essential to the MZT because it is absent in mouse two- cell embryos. Our results for HMGB are analogous to those obtained from the mouse. If we take into account the timing of MZT, we would expect very low HMGB1 protein levels in bovine eight-cell embryos. Thus, the role of HMGB1 in the embryo still remains to be elucidated and it could mainly act in pre-MZT embryos. The results presented here show, for the first time, the expression pattern of NFAR in mammalian preimplantation development. However, it must be specified that the pattern of expression illustrated here combine both NFAR-1 and NFAR-2 mRNA (Fig. 3). Nevertheless, if the sharp decrease in NFAR transcript level observed in eight-cell embryos (Fig. 3) is due to translation and subsequent degradation, this would suggest that NFAR is also implicated in the MZT. Furthermore, it would be very interesting to examine the possibility that NFAR is related to mRNA masking in oocytes and early embryos up until the MZT, as has been found in Xenopus [51]. In the case of TEAD2, a previous publication has shown that this factor's mRNA is associated with polysomes in the two-cell mouse embryo, but not in the oocyte or in the one- cell embryo [52]. This strongly suggests that TEAD2 mRNA is translated at this stage to coincide with the MZT. Translation at this point seems to cause the loss of TEAD2 transcript in mouse two-cell embryos. In the bovine, as in the mouse, TEAD2 mRNA is present at all stages of embryonic development before MZT. The decrease of its mRNA level at the four- and eight-cell stages suggests that it could be translated to provide TEAD2 proteins for the MZT [52]. As for OCT-4, our results coincide with those of another study showing mRNA expression patterns in bovine preimplantation embryos, thereby confirming the accuracy of our technique [53]. As previously demonstrated, the OCT-4 protein is present in the bovine preimplantation embryos nucleus at all stages, which suggests that it most likely plays a role in the transcription of many genes important for early embryo development [33].

ATF-1 has a quite fascinating expression pattern. Contrary to other genes presented in this study, ATF-1 expression pattern shows no significant decrease at the eight-cell stage compared with previous stages of development (Fig. 4). If that mRNA present in the eight-cell embryos is from maternal origin, it would suggest that it is important for the MZT because its mRNA has been preserved until that stage. Alternatively, if this is due to a new embryonic transcript, it is still intriguing because it would be one of the earliest embryonic transcription factors to be expressed in bovine embryos and therefore plays a primary role in the MZT.

Finally, for all the genes studied here, the genes presented in Figure 5 are the only ones that demonstrate a low level of mRNA at the blastocyst stage compared with other stages. In fact, the mRNA levels of these four genes is higher in the oocyte than in all other stages and do not increase at the blastocyst stage after the genome activation. This strongly suggests an important role for these factors in the very first developmental stages of bovine embryos. Earlier expression studies with HMGN2 in mouse embryos showed that its mRNA is present in high quantities in the mouse GV oocyte. Then the level decreases until the two- cell stage (at the MZT) and increases from the four-cell embryo to the blastocyst stages [27]. Our results show a similar expression pattern in bovine embryos, in which the lowest level is detectable at the MZT. However, unlike for the mouse, there is not a significant increase after the MZT, according to the mRNA level observed in blastocysts. Our HMGN2 expression pattern is very similar to that of the HMGN1 except that no increase is observed at the blastocyst stage. Consequently, our results suggest a possible implication for HMGN2 in early bovine development and genome activation. In previous studies, MSY2 protein has been detected in mouse oocytes and one-cell embryos. Yet no MSY2 protein has been detectable in late two-cell embryos, the time at which major mRNA degradation occurs and maternal mRNA is replaced by new embryonic mRNA [36, 38]. The MSY2 mRNA level follows roughly the same pattern as the protein level except that a slight mRNA presence is still detected in the two-cell embryos and even smaller amounts in blastocysts [36]. This could indicate that MSY2 is present in pre-MZT mouse embryos to protect maternal mRNA from degradation until it is translated. The results observed here are consistent with those of previous studies because we detect MSY2 in stages leading up to MZT, when mRNA protection is needed and the amount of MSY2 decreases around the MZT. At this point, stored maternal mRNA has to be translated to produce essential proteins for further development. The disappearance of MSY2 coincides with mRNA degradation of almost all genes examined in this study. This supports the idea that the mRNA is protected from degradation up until the time it needs to be recruited by the translation machinery to produce the required proteins, after which it is degraded. The remaining factor TBP has a mRNA expression pattern that has been previously characterized in mouse embryo development [52]. This study showed that the highest TBP mRNA level is observed in GV oocytes. This level then first decreases during maturation and then again during the first third of the two-cell stage [52]. Protein localization experiments have shown that, in the mouse, TBP nuclear localization increases after fertilization up until the two-cell stage embryos [54]. Our results support the findings that the mRNA level decreases from the oocyte stage until the MZT. The results presented by these previous studies confirm that protein level does not necessarily follow the corresponding mRNA level and that protein accumulation may correlate with the mRNA diminution [52, 54, 55]. Our results consequently suggest that the TBP protein tends to accumulate in the pre-MZT embryo. This is supported by the slight level of transcription observed in pre-MZT bovine embryos [6]. Pre-MZT transcription requires TBP because transcription in pre-MZT embryos is regulated by TATA-box promoters, as shown in the mouse [56–58]. The decrease in TBP transcript level observed between the four- to eight- cell stages suggests the production of new proteins and coincides with the timing of the MZT when the use of TATA- box promoters is replaced by the use of TATA-less promoters in mouse [56, 57] and in human embryos [59]. However, the use of TATA-less promoters does not mean that the embryo is completely independent of TBP during this period because it has been shown that TBP may still be required for TATA-less promoter activity [60]. The large amount of TBP mRNA found in oocytes, compared with other stages including the blastocyst, is consistent with expression patterns of other species [52, 55] and reinforces the perceived importance of TBP for basal transcription in pre-MZT bovine embryos.

We have demonstrated that the 15 genes examined in this study are all present in bovine oocytes throughout pre- MZT embryonic development in their mRNA form. These results support the hypothesis that these factors could be implicated in the activation of embryonic transcription. It also lends support to the idea that transcription factors may be found as maternally stored mRNA in the oocyte until their recruitment for translation just in time for MZT. Despite previous results, where DNA replication and chromatin regulation have been suggested as a limiting step in activation of embryonic transcription, the present study raises the possibility that transcription factors could be rate limiting. Furthermore, these results open the door to new avenues of research by proposing novel transcription factors that could be directly implicated in the MZT.


   ACKNOWLEDGMENTS
 
I thank Dr. Atef Ali and Karine Tremblay for their technical assistance. I also acknowledge Dr. Robert Viger for his critical reading of this manuscript. Finally, special thanks to Dr. Susan Novak for her help in the statistical analysis and to Dr. Michael K. Dyck for correcting the manuscript.


   FOOTNOTES
 
1 Supported by the Canada Research Chair and the Natural Science and Engineering Research Council of Canada. Back

2 Correspondence. FAX: 418 656 3766;marc-andre.sirard{at}crbr.ulaval.ca Back

Received: 5 September 2003.

First decision: 4 October 2003.

Accepted: 19 January 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Kanka J. Gene expression and chromatin structure in the preimplantation embryo. Theriogenology 2003 59:3-19[CrossRef][Medline]
  2. Telford NA, Watson AJ, Schultz GA. Transition from maternal to embryonic control in early mammalian development: a comparison of several species. Mol Reprod Dev 1990 26:90-100[CrossRef][Medline]
  3. Schultz RM. Regulation of zygotic gene activation in the mouse. Bioessays 1993 15:531-538[CrossRef][Medline]
  4. Etkin LD, Balcells S. Transformed Xenopus embryos as a transient expression system to analyze gene expression at the midblastula transition. Dev Biol 1985 108:173-178[CrossRef][Medline]
  5. Memili E, First NL. Control of gene expression at the onset of bovine embryonic development. Biol Reprod 1999 61:1198-1207[Abstract/Free Full Text]
  6. Memili E, First NL. Zygotic and embryonic gene expression in cow: a review of timing and mechanisms of early gene expression as compared with other species. Zygote 2000 8:87-96[CrossRef][Medline]
  7. Schultz RM, Davis W Jr, Stein P, Svoboda P. Reprogramming of gene expression during preimplantation development. J Exp Zool 1999 285:276-282[CrossRef][Medline]
  8. Marmorstein R. Protein modules that manipulate histone tails for chromatin regulation. Nat Rev Mol Cell Biol 2001 2:422-432[CrossRef][Medline]
  9. Stein P, Worrad DM, Belyaev ND, 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]
  10. 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]
  11. McGraw S, Robert C, Massicotte L, Sirard MA. Quantification of histone acetyltransferase and histone deacetylase transcripts during early bovine embryo development. Biol Reprod 2003 68:383-389[Abstract/Free Full Text]
  12. Bianchi ME, Beltrame M. Upwardly mobile proteins. Workshop: the role of HMG proteins in chromatin structure, gene expression and neoplasia. EMBO Rep 2000 1:109-114[CrossRef][Medline]
  13. Woychik NA, Hampsey M. The RNA polymerase II machinery: structure illuminates function. Cell 2002 108:453-463[CrossRef][Medline]
  14. Nikolov DB, Burley SK. RNA polymerase II transcription initiation: a structural view. Proc Natl Acad Sci U S A 1997 94:15-22[Abstract/Free Full Text]
  15. Lee JS, Galvin KM, Shi Y. Evidence for physical interaction between the zinc-finger transcription factors YY1 and Sp1. Proc Natl Acad Sci U S A 1993 90:6145-6149[Abstract/Free Full Text]
  16. Donohoe ME, Zhang X, McGinnis L, Biggers J, Li E, Shi Y. Targeted disruption of mouse Yin Yang 1 transcription factor results in peri- implantation lethality. Mol Cell Biol 1999 19:7237-7244[Abstract/Free Full Text]
  17. Reeves R, Beckerbauer L. HMGI/Y proteins: flexible regulators of transcription and chromatin structure. Biochim Biophys Acta 2001 1519:13-29[Medline]
  18. Robert C, Barnes FL, Hue I, Sirard MA. Subtractive hybridization used to identify mRNA associated with the maturation of bovine oocytes. Mol Reprod Dev 2000 57:167-175[CrossRef][Medline]
  19. Nakamura Y, Moriuchi R, Nakayama D, Yamashita I, Higashiyama Y, Yamamoto T, Kusano Y, Hino S, Miyamoto T, Katamine S. Altered expression of a novel cellular gene as a consequence of integration of human T cell lymphotropic virus type 1. J Gen Virol 1994 75:2625-2633[Abstract/Free Full Text]
  20. Vo N, Goodman RH. CREB-binding protein and p300 in transcriptional regulation. J Biol Chem 2001 276:13505-13508[Free Full Text]
  21. Hai T, Hartman MG. The molecular biology and nomenclature of the activating transcription factor/cAMP responsive element binding family of transcription factors: activating transcription factor proteins and homeostasis. Gene 2001 273:1-11[CrossRef][Medline]
  22. Andrisani OM. CREB-mediated transcriptional control. Crit Rev Eukaryot Gene Expr 1999 9:19-32[Medline]
  23. De Cesare D, Fimia GM, Sassone-Corsi P. Signaling routes to CREM and CREB: plasticity in transcriptional activation. Trends Biochem Sci 1999 24:281-285[CrossRef][Medline]
  24. Yagi R, Chen LF, Shigesada K, Murakami Y, Ito Y. A WW domain- containing yes-associated protein (YAP) is a novel transcriptional co- activator. Embo J 1999 18:2551-2562[CrossRef][Medline]
  25. Vassilev A, Kaneko KJ, Shu H, Zhao Y, DePamphilis ML. TEAD/ TEF transcription factors use the activation domain of YAP65, a Src/ Yes-associated protein localized in the cytoplasm. Genes Dev 2001 15:1229-1241[Abstract/Free Full Text]
  26. Bustin M, Trieschmann L, Postnikov YV. The HMG-14/-17 chromosomal protein family: architectural elements that enhance transcription from chromatin templates. Semin Cell Biol 1995 6:247-255[CrossRef][Medline]
  27. Mohamed OA, Bustin M, Clarke HJ. High-mobility group proteins 14 and 17 maintain the timing of early embryonic development in the mouse. Dev Biol 2001 229:237-249[CrossRef][Medline]
  28. Bianchi ME, Beltrame M. Flexing DNA: HMG-box proteins and their partners. Am J Hum Genet 1998 63:1573-1577[CrossRef][Medline]
  29. Ovsenek N, Karn HA, Heikkila JJ. Analysis of CCAAT box transcription factor binding activity during early Xenopus laevis embryogenesis. Dev Biol 1991 145:323-327[CrossRef][Medline]
  30. Saunders LR, Jurecic V, Barber GN. The 90- and 110-kDa human NFAR proteins are translated from two differentially spliced mRNAs encoded on chromosome 19p13. Genomics 2001 71:256-259[CrossRef][Medline]
  31. Saunders LR, Perkins DJ, Balachandran S, Michaels R, Ford R, Mayeda A, Barber GN. Characterization of two evolutionarily conserved, alternatively spliced nuclear phosphoproteins, NFAR-1 and -2, that function in mRNA processing and interact with the double-stranded RNA-dependent protein kinase, PKR. J Biol Chem 2001 276:32300-32312[Abstract/Free Full Text]
  32. Ovitt CE, Scholer HR. The molecular biology of Oct-4 in the early mouse embryo. Mol Hum Reprod 1998 4:1021-1031[Abstract/Free Full Text]
  33. van Eijk MJ, van Rooijen MA, Modina S, Scesi L, Folkers G, van Tol HT, Bevers MM, Fisher SR, Lewin HA, Rakacolli D, Galli C, de Vaureix C, Trounson AO, Mummery CL, Gandolfi F. Molecular cloning, genetic mapping, and developmental expression of bovine POU5F1. Biol Reprod 1999 60:1093-1103[Abstract/Free Full Text]
  34. Saijoh Y, Fujii H, Meno C, Sato M, Hirota Y, Nagamatsu S, Ikeda M, Hamada H. Identification of putative downstream genes of Oct-3, a pluripotent cell-specific transcription factor. Genes Cells 1996 1:239-252[Abstract]
  35. Kaneko KJ, Cullinan EB, Latham KE, DePamphilis ML. Transcription factor mTEAD-2 is selectively expressed at the beginning of zygotic gene expression in the mouse. Development 1997 124:1963-1973[Abstract]
  36. Yu J, Hecht NB, Schultz RM. Expression of MSY2 in mouse oocytes and preimplantation embryos. Biol Reprod 2001 65:1260-1270[Abstract/Free Full Text]
  37. Yu J, Hecht NB, Schultz RM. RNA-binding properties and translation repression in vitro by germ cell-specific MSY2 protein. Biol Reprod 2002 67:1093-1098[Abstract/Free Full Text]
  38. Gu W, Tekur S, Reinbold R, Eppig JJ, Choi YC, Zheng JZ, Murray MT, Hecht NB. Mammalian male and female germ cells express a germ cell-specific Y-Box protein, MSY2. Biol Reprod 1998 59:1266-1274[Abstract/Free Full Text]
  39. Buratowski S. Snapshots of RNA polymerase II transcription initiation. Curr Opin Cell Biol 2000 12:320-325[CrossRef][Medline]
  40. Holm P, Booth PJ, Schmidt MH, Greve T, Callesen H. High bovine blastocyst development in a static in vitro production system using SOFaa medium supplemented with sodium citrate and myo-inositol with or without serum-proteins. Theriogenology 1999 52:683-700[CrossRef][Medline]
  41. Ali A, Sirard MA. Effect of the absence or presence of various protein supplements on further development of bovine oocytes during in vitro maturation. Biol Reprod 2002 66:901-905[Abstract/Free Full Text]
  42. Rozen S, Skaletsky HJ. Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds.), Bioinformatics Methods and Protocols: Methods in Molecular Biology. Totowa, NJ: Humana Press, 2000:365–386 (code available at http://www.genome.wi.mit.edu/genome_software/other/primer3.html)
  43. Robert C, McGraw S, Massicotte L, Pravetoni M, Gandolfi F, Sirard MA. Quantification of housekeeping transcript levels during the development of bovine preimplantation embryos. Biol Reprod 2002 67:1465-1472[Abstract/Free Full Text]
  44. Lonergan P, Gutierrez-Adan A, Rizos D, Pintado B, de la Fuente J, Boland MP. Relative messenger RNA abundance in bovine oocytes collected in vitro or in vivo before and 20 hr after the preovulatory luteinizing hormone surge. Mol Reprod Dev 2003 66:297-305[CrossRef][Medline]
  45. Lonergan P, Rizos D, Gutierrez-Adan A, Moreira PM, Pintado B, de la Fuente J, Boland MP. Temporal divergence in the pattern of messenger RNA expression in bovine embryos cultured from the zygote to blastocyst stage in vitro or in vivo. Biol Reprod 2003 69:1424-1431[Abstract/Free Full Text]
  46. Tomek W, Torner H, Kanitz W. Comparative analysis of protein synthesis, transcription and cytoplasmic polyadenylation of mRNA during maturation of bovine oocytes in vitro. Reprod Domest Anim 2002 37:86-91[CrossRef][Medline]
  47. Grosset C, Chen CY, Xu N, Sonenberg N, Jacquemin-Sablon H, Shyu AB. A mechanism for translationally coupled mRNA turnover: interaction between the poly(A) tail and a c-fos RNA coding determinant via a protein complex. Cell 2000 103:29-40[CrossRef][Medline]
  48. Hosoda N, Kobayashi T, Uchida N, Funakoshi Y, Kikuchi Y, Hoshino S, Katada T. Translation termination factor eRF3 mediates mRNA decay through the regulation of deadenylation. J Biol Chem 2003 278:38287-38291[Abstract/Free Full Text]
  49. Thompson EM, Legouy E, Christians E, Renard JP. Progressive maturation of chromatin structure regulates HSP70.1 gene expression in the preimplantation mouse embryo. Development 1995 121:3425-3437[Abstract]
  50. Spada F, Brunet A, Mercier Y, Renard JP, Bianchi ME, Thompson EM. High mobility group 1 (HMG1) protein in mouse preimplantation embryos. Mech Dev 1998 76:57-66[CrossRef][Medline]
  51. Brzostowski J, Robinson C, Orford R, Elgar S, Scarlett G, Peterkin T, Malartre M, Kneale G, Wormington M, Guille M. RNA-dependent cytoplasmic anchoring of a transcription factor subunit during Xenopus development. Embo J 2000 19:3683-3693[CrossRef][Medline]
  52. Wang Q, Latham KE. Translation of maternal messenger ribonucleic acids encoding transcription factors during genome activation in early mouse embryos. Biol Reprod 2000 62:969-978[Abstract/Free Full Text]
  53. Daniels R, Hall V, Trounson AO. Analysis of gene transcription in bovine nuclear transfer embryos reconstructed with granulosa cell nuclei. Biol Reprod 2000 63:1034-1040[Abstract/Free Full Text]
  54. 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]
  55. Veenstra GJ, Destree OH, Wolffe AP. Translation of maternal TATA- binding protein mRNA potentiates basal but not activated transcription in Xenopus embryos at the midblastula transition. Mol Cell Biol 1999 19:7972-7982[Abstract/Free Full Text]
  56. Davis W Jr, Schultz RM. Developmental change in TATA-box utilization during preimplantation mouse development. Dev Biol 2000 218:275-283[CrossRef][Medline]
  57. Majumder S, DePamphilis ML. TATA-dependent enhancer stimulation of promoter activity in mice is developmentally acquired. Mol Cell Biol 1994 14:4258-4268[Abstract/Free Full Text]
  58. Nothias JY, Majumder S, Kaneko KJ, DePamphilis ML. Regulation of gene expression at the beginning of mammalian development. J Biol Chem 1995 270:22077-22080[Free Full Text]
  59. 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]
  60. Pugh BF, Tjian R. Transcription from a TATA-less promoter requires a multisubunit TFIID complex. Genes Dev 1991 5:1935-1945[Abstract/Free Full Text]



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