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Embryo |
Centre de Recherche en Biologie de la Reproduction, Département des Sciences Animales, Université Laval,Québec, Canada G1K 7P4
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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early development, embryo, gene regulation, in vitro fertilization, oocyte development
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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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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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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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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).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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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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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 418 656 3766;marc-andre.sirard{at}crbr.ulaval.ca
Received: 5 September 2003.
First decision: 4 October 2003.
Accepted: 19 January 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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