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Regular Article |
a Department of Obstetrics and Gynaecology, Queen Mary Hospital, and
b Department of Zoology, The University of Hong Kong, Hong Kong, China
ABSTRACT
The objectives of this study were to compare the mRNA expression patterns in early mouse embryos in different culture conditions by differential display reverse transcription-polymerase chain reaction (DDRT-PCR). Embryos developed in vivo, cultured in vitro, and cocultured with human oviductal epithelial cells were studied at the 2-cell, 4-cell, 8-cell/morula, and blastocyst stages. Messenger RNA profiles were displayed by DDRT-PCR using downstream T11VV (V = A, C, or G) and upstream decamer primers. Total cDNA banding patterns were highly conserved in the three groups studied. Some fragments are unique in different culture conditions. Thirteen out of the 40 selected differentially expressed clones were characterized. The DNA sequence analyses of these clones displayed high sequence homology with cDNA sequences in the mouse expressed sequence tag database. Using semiquantitative RT-PCR, we confirmed differential expression of these DD amplicons in the three groups of embryos. The temporal expression of some of the selected DD amplicons during preimplantation development were studied in the three groups of embryos. In conclusion, DDRT-PCR is an effective tool for contrasting gene expression patterns and characterizing mRNA transcripts in mouse embryo.
fertilization, gene regulation, oviduct
INTRODUCTION
Mammalian preimplantation embryo development involves cell proliferation and differentiation. Fertilization triggers the completion of meiotic division in the oocyte and induces the subsequent embryonic development. This latter process involves degradation of the maternal RNAs and proteins and the activation of the embryonic genome in the developing embryos. Detailed control mechanisms on these processes are still unclear. It has been proposed that embryonic genome activation starts at the 2-cell stage in mouse embryos [1]. Early genes such as eIF-1A [2], U2afbp-rs [3], and hsp70.1 [4] may play important roles in embryonic genome activation. While the detailed mechanism of genome activation is lacking, the acquisition of a transcriptionally repressive environment and changes in chromatin structure by alteration of histone deacetylase activity can block or stimulate repression of markers of genome activation [2, 5]. Apart from genes that are involved in embryonic genome activation, gene expression that is specific to embryos at certain cleavage stages of development has also been identified [6].
Messenger RNA differential display (DD) is a very sensitive reverse transcription-polymerase chain reaction (RT-PCR)-based method [7] that would easily allow comparisons of two or more mRNA samples prepared from small amount of tissues. It is particularly suitable for developmental studies involving temporal changes in gene expression in embryos. Using this method, genes that are temporally and differentially expressed in mouse embryos [6, 8] have been isolated for characterization.
Embryos cultured in vitro have a blastulation rate lower than those developed in vivo [9]. This can be explained by the suboptimal culture environment, lack of growth factors/embryotrophic factors, and presence of toxic substances in the culture medium. Various studies [10–14], including those from this laboratory [15–17], have shown that coculture improves the morphological development and implantation of the treated embryos, partly by the production of embryotrophic factors from the oviductal cells [18]. However, the detailed molecular mechanism by which coculture improves embryo development in vitro is not at all clear.
We hypothesize that coculture alters embryonic gene expression and thereby improves embryo development. To test this hypothesis, we adopted the DD technique to compare the gene expression profiles of preimplantation mouse embryos with and without coculture. Differentially expressed genes were identified and confirmed with semiquantitative RT-PCR. The identities of these genes will be of interest and essential to delineate the molecular changes during early embryonic development. It is well known that in vitro culture produces artifacts in embryo development such as a 2-cell block. Coculture is known to improve embryo development and to eliminate partially or completely some of the known artifacts, e.g., developmental blockage and fragmentation. Whether cocultured embryos behave similarly to those embryos developed naturally in the maternal body is unknown. To address this question we compared the gene expression profiles between the cocultured embryos and those flushed directly from the reproductive tract at different gestational ages.
MATERIALS AND METHODS
Oocyte and Embryo Collection and Culture
Murine oocytes and embryos were collected from 6- to 8-wk-old BALB/c male x MF-1 female mice and cultured in Chatot-Ziomek-Bavister (CZB) medium as described elsewhere [19]. In brief, embryos were cultured or cocultured with human oviductal cells in CZB medium for the first 48 h and in CZB medium supplemented with glucose for the rest of the culture. Our unpublished data showed that embryos from this strain of mouse grew just as well in CZB medium as in potassium simplex optimized medium (KSOM), the other medium commonly used for embryo culture. In vivo-developed mouse embryos at the 2-cell, 4-cell, 8-cell/morula, and blastocyst stages were flushed from either oviducts or uteri at 42–46, 67–71, 88–92, and 100–104 h post-hCG, respectively. The in vitro-cultured and cocultured embryos [16] were evaluated by morphological observation under an inverted microscope. Embryos at the 2-cell, 4-cell, 8-cell/morula, and blastocyst stages were collected at 46–48, 72–74, 96–100, and 118–122 h post-hCG, respectively.
Extraction of RNA
Messenger RNAs from mouse embryos were extracted by Dynabeads mRNA Direct Kit (Dynal AS, Oslo, Norway) according to the manufacturer's protocol. In brief, the zona pellucida of mouse embryos was dissolved by acid Tyrode (Sigma, St. Louis, MO) treatment [20]. Fifteen to 40 embryos were transferred with minimal volume of medium into 300 µl of lysis buffer (100 mM Tris-HCl; pH 7.5, 500 mM LiCl, 10 mM EDTA, 1% lithium dodecylsulfate [LiDS], and 5 mM dithiothreitol) and mixed with 10 µl of Dynabeads oligo(dT)25 for 3–5 min. The bound mRNAs were washed twice in washing buffer containing LiDS (10 mM Tris-HCl; pH 7.5, 0.15 M LiCl, 1 mM EDTA, and 0.1% LiDS), then three times in washing buffer (10 mM Tris-HCl; pH 7.5, 0.15 M LiCl, 1 mM EDTA). Messenger RNAs were eluted with 10–20 µl of diethyl pyrocarbonate (DEPC)-treated water.
Messenger RNA DD
Complementary DNAs were synthesized using the First-Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer's protocol. Embryonic mRNA was first denatured at 65°C for 10 min, then 2.5 µM anchor primer (dT11VV, where V = A, C, or G) and 5 µl of bulk first-strand cDNA reaction mixes were added. The samples were further incubated at 37°C for 1 h, followed by incubation at 95°C for 5 min to inactivate the reverse transcriptase.
Differential mRNA display reactions were performed as described in the Differential Display Kit protocol (Display Systems Biotech, Vista, CA). Polymerase chain reactions were performed in 1x PCR buffer (10 mM Tris-HCl, pH .3, 50 mM KCl and 1.5 mM MgCl2), 2 µM dNTP, 0.5 µM arbitrary decamer, 2.5 µM anchor primer, 0.1 µl [
-33P]dATP (3000 Ci/mmol; Dupont NEN, Boston, MA), 0.5 U Taq DNA polymerase (Roche Molecular Biochem., Indianapolis, IN) and 2 µl of first-strand cDNA. The cDNA sequences were amplified in a Perkin-Elmer model 480 thermocycler (Perkin-Elmer Applied Biosys, Foster City, CA) using the following profile: 94°C for 30 sec, 40°C for 90 sec, and 72°C for 60 sec. After 40 cycles of PCR, the reactions were incubated at 72°C for an additional 5 min. The amplified products were separated in a 6% denaturing polyacrylamide gel [21]. The gel was dried, and autoradiography was carried out using Kodak BioMax MR film (Eastman Kodak, Rochester, NY).
Cloning and Sequencing of mRNA DD Amplicons
Differential expressed bands were excised from the gel, heated at 95°C in 0.5 ml H2O for 10 min. The gel was removed from each PCR tube, and the supernatant was used for PCR reamplification. The PCR conditions were similar to DDRT-PCR protocols, except that a 42°C annealing temperature was used and [-33P]dATP was substituted with unlabeled dATP. Reamplified amplicons were cloned into pGEM-T easy vector (Promega Corp., Madison, WI) following the manufacturer's instructions and sequenced with an ABI 310 genetic analyzer (Perkin-Elmer) using the dRhodamine Terminator Cycle Sequencing Kit (Perkin-Elmer) with T7 and SP6 primers. The sequences were assembled using the DNasis version 2.1 program (Hitachi Software Engineering, San Bruno, CA) and analyzed using the online computer BLAST program [22].
Semiquantitative RT-PCR Assay
The relative changes of mRNA transcripts were determined using a semiquantitative RT-PCR assay as described [23]. In brief, mRNAs from 10 mouse embryos were isolated using Dynabeads mRNA Direct Kit (Dynal AS) as described above and resuspended in DEPC-treated water to obtain equivalent embryo poly(A)+ at each stage [24]. The mRNAs were subjected to semiquantitative RT-PCR using the one-tube Access RT-PCR System (Promega). One microliter of mRNA was reverse transcribed and PCR amplified in 50 µl of reaction volume containing 1x avian myeloblastosis virus (AMV)/thermus flavus (Tfl) buffer, 5 U AMV reverse transcriptase, 5 U Tfl DNA polymerase, 1 mM MgSO4, 200 µM dNTP, and 1 µM gene-specific primers (Table 1). The reverse transcription was carried out at 48°C for 45 min, then followed by 2 min at 94°C to inactivate the AMV reverse transcriptase. The PCR conditions were 94°C for 30 sec, 60°C for 1 min, and 68°C for 2 min for 28–40 cycles. For normalization of gene amplification, a constant amount of exogenous human ß-globin RNA was added into each PCR reaction. The ß-globin was synthesized using the Maxiscript T7 transcription kit (Ambion Inc., Austin, TX) and quantified by UV spectrophotometry. The PCR products were resolved in 2% NuSieve 3:1 agarose (FMC BioProducts, Rockland, MN). The images were quantified by densitometric scanning followed by ImageQuant version 3.3 (Molecular Dynamics, Sunnyvale, CA) analysis. Intensities of the signals were normalized to those of ß-globin products as ratios to produce arbitrary units of relative abundance. Each mRNA transcript was assayed for a minimum of three independent batches of in vivo-developed, in vitro-cultured, and cocultured mouse embryos. The mean values and their accompanying standard error were reported.
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Statistical Analysis
Data were analyzed using the SigmaStat (Jandel Scientific, San Rafael, CA) software package. Analysis of differences in the means between two or more populations was tested using a one-way ANOVA followed by multiple pairwise comparisons using a Student-Newman-Keuls method. Differences of P 
0.05 were considered significant.
RESULTS
Identification of Differentially Expressed Genes from Mouse Embryos in Different Culture Conditions
Differential display analysis allows the observation of altered gene expression and subsequent isolation of cDNAs that correspond to mRNAs in developing mouse embryos [6]. To identify genes expressed in different culture conditions, we compared the mRNA expression profiles of mouse embryos developed in vivo, cultured in vitro, and cocultured with human oviductal cells. Mouse embryos at four developmental stages (2-cell, 3- to 4-cell, morula, and blastocyst) were collected. Messenger RNAs from morula stage embryos were extracted and subjected to DD analysis. A representative example of this analysis was given in Figure 1. By using a combination of six arbitrary decamer primers and three two-base anchored oligo(dT) primers (dT11VV, where V = A, C, or G), we generated about 800 PCR amplicons, 40 of them were differentially displayed in different culture conditions. The DD patterns were very reproducible between different batches of embryos at the same stage of development. The majority of amplicons from an embryonic stage were common to the three developmental conditions being studied.
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Purification and Sequencing of cDNAs from Heterogenous Amplicon Mixes
The differentially expressed bands were excised and reamplified. The reamplified DD amplicons were cloned into pGEM-T easy vector. Both DNA strands of the DD amplicons were sequenced using the T7 and SP6 primers complementary to the vector sequences. The sequences of the DD amplicons were compared with the DNA sequences of the GenBank/EMBL database. The expression patterns and the identities of 14 amplicons were summarized in Table 2. Nine of them shared similarity with mouse cDNA clones without known functions (clones 13, 17, 25, 37, 41, 121, 151, 173, and 192). Clone 21 was identical to clone 112. Clone 21 (112), 29, 33, and 144 showed high homology with mouse ezrin (99%), eIF-1A (84%), BAC clone AC005259 (94%), and ring1B (97%), respectively. Interestingly, clone 41 was highly homologous (95%) to the 3'-end of human E3KARP (accession no. AF004900), an NHE3 Na+/H+ exchanger regulatory protein that might interact with cytoskeletal protein ezrin (clones 21 and 112).
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Confirmation of DD Analysis by Semiquantitative RT-PCR
To confirm the results of the differential mRNA display analysis, mRNAs isolated from the mouse embryos were subjected to semiquantitative RT-PCR analysis. The primer sequences and the number of PCR cycles used for different DD amplicons were summarized in Table 1. On the whole, the expression patterns of the DD amplicons as determined by semiquantitative RT-PCR patterns were fairly similar to that found by the DD. Figure 2 showed one of the representative comparisons using embryos at morula stage. Among the three groups of embryos, clones 13, 21, 25, 41, 144, and 192 were expressed more in in vivo-developed embryos. Higher expression of clones 29, 33, and 37 in in vitro-cultured embryos and clones 29, 37, and 151 in cocultured mouse embryos were found when compared with embryos developed in vivo.
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Differential Expression of DD Amplicons During Embryonic Development
The relative changes of mRNA transcripts for DD amplicons with known functions (clones 21, 29, 41, and 144) and two expressed sequence tag (EST; clones 151 and 173) were determined using a semiquantitative RT-PCR assay. To normalize the RT-PCR reaction efficiency, an exogenously added ß-globin mRNA was used as an internal standard [25]. This assay was used to compare the relative representation of one mRNA among different samples but not the absolute representation of the mRNA to that of another.
We observed similar amplification efficiency of the internal standard, ß-globin gene in all the samples tested (Fig. 3). Expression levels of the ß-actin gene and ezrin (clone 21) increased during development among the three developmental conditions. The transcription factor, eIF-1A (clone 29), was activated at the 2-cell stage but decreased at the 4-cell stage and increased again at the morula stage. The levels of E3KARP (clone 41) expression were fairly constant in the cultured embryo with an increasing trend at the morula stage (P < 0.05). However, there was a significant increase in E3KARP expression in the in vivo-developed morula embryos when compared with that in oocytes (P < 0.05). The expression profile of clones 144 and 151 in in vivo-developed embryos showed a decreasing trend from the 2-cell stage to the blastocyst stage, whereas that of cocultured embryos remained constant until the blastocyst, at which the level of expression dropped. On the other hand, expression of clone 151 in medium alone-cultured embryos increased gradually until the morula stage (P < 0.05), after which its expression decreased. However, the expression profiles of clone 173 varied in different cultured embryos. The in vitro-cultured embryos remained unchanged throughout development. In the cocultured embryos, its level increased rapidly at the 4-cell stage up to the morula and decreased thereafter. Interestingly, the expression profile of clone 173 in in vivo-developed embryos was similar to that of clone 29, i.e., expression increased at the 2-cell stage but decreased at the 4-cell stage and increased again at the morula stage.
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DISCUSSION
The DD technique was first developed by Liang and Pardee [7] and has been used to study gene expression patterns in developing embryos. It has been used to detect differential expression of mitochondrial 16S rRNA in p53-deficient mouse embryos [8] and to study the gene expression of mouse embryos developed in vivo [6]. Using this technique, we generated DD profiles of mouse embryos developed in vivo, cultured in vitro, and cocultured with human oviductal cells. The majority of the DD bands are common among the three groups of embryos. This is not surprising as these may represent genes with housekeeping functions. The DD amplicons that were differentially expressed in these embryos were selected and confirmed by semiquantitative RT-PCR assay. By using the same amount of poly(A)+ RNA for mouse embryos at different stages [24], we quantified the expression levels of 13 selected DD amplicons using RT-PCR (Fig. 2).
The DNA sequence analyses revealed that most of the amplicons (clones 13, 17, 25, 37, 41, 121, 151, 173, and 192) shared high homology with cDNA sequences in the mouse EST database (Table 2). However, little is known about the functions of these genes in early embryonic development of the mouse. From the results of semiquantitative RT-PCR, the expression levels of some DD amplicons (clones 25 and 192) were higher in oocytes, suggesting that these transcripts may be of maternal origin and down-regulated during development (data not shown).
It is generally believed that the oviduct provides the best microenvironment for early embryonic development, therefore in vivo-developed embryos should have the best developmental potential. Based on this assumption, coculture with oviductal cells is intended to simulate the in vivo developmental condition. Comparing the DD profiles between embryos in the three developmental conditions (Fig. 1 and unpublished observation), the gene expression pattern of cocultured embryos is more similar to that of in vitro-cultured embryos than to the in vivo-developed embryos, suggesting that the present coculture system is not yet optimal.
There are several possible explanations that may account for the inferior development of embryos even under an oviductal cell coculture system when compared with the in vivo microenvironment. First, the cocultured cells are competing with the embryos for resources in a coculture system. The depletion of nutrients as well as generation of metabolic waste by the cocultured cells may decrease the efficiency of embryotrophic factors in stimulating embryo development. Second, embryo culture medium is used in coculture. The oviductal cells may not be at their best performance when being cultured in such medium. Third, cultured oviductal cells behave differently from those found in vivo. Fourth, our unpublished data show that the embryotrophic factors from mouse oviductal cells are different from that of human oviductal cells. Thus, it is possible that the latter are less effective in stimulating mouse embryo development.
Despite the similarity in mRNA expression profiles between the medium alone-cultured embryos and the cocultured embryos, differences in the profiles are easily noticeable. Two of them (clones 151 and 173) have been determined semiquantitatively at different embryonic developmental stages in the present study (Fig. 3). It is known that gene expression in embryos is affected by different culture environments [26] and that mouse embryos can develop well in vitro in different culture media [27]. Whether the differences in gene expression between the cocultured and the medium alone-cultured embryos are responsible for the better development of the former remains to be investigated.
The transition from the maternal to embryonic genome control in early mammlian embryo is not fully understood. Evidence suggests that acquisition of a transcriptionally repressive environment and changes in chromatin structure by alteration of histone deacetylase activity can block or stimulate repression of markers of genome activation [2, 5]. Interestingly, clone 144 is highly homologous to mouse ring1B, a ring1A homolog that interacts with M33, a member of polycomb group that acts as chromatin-associated multimeric protein complexes and has been implicated in mesoderm patterning in mouse development [28]. More importantly, the ring1A protein can function as repressor when tethered to promoters by means of a DNA binding domain located at the N-terminal of the protein. The high expression of ring1B in mouse oocyte and its subsequent decrease with further development in vivo may relieve the repressed state of mouse embryos during development. In this connection, the developmental potential of medium alone-cultured embryos is lowest among the three groups, and coincidentally the expression of clone 144 remains high throughout development (Fig. 3). Nevertheless, the role of ring1B on early embryonic development is still unknown.
Using a semiquantitative RT-PCR assay, eukaryotic translation initiation factor eIF-1A was observed to increase transiently at the 2-cell stage in mouse embryos [2, 25]. This transient expression of eIF-1A is coincidentally associated with the time when embryonic gene activation is proposed to occur [25]. The present study confirmed these reports and further demonstrated that a similar pattern of eIF-1A (clone 29) expression was found in cocultured as well as medium alone-cultured embryos. It also strengthens the hypothesis that the transient expression of eIF-1A in mammalian embryos is a conserved pattern of gene expression associated with embryonic genome activation [25]. Thus, an understanding of eIF-1A gene regulation and function may shed light on the isolation of related genes that are involved in embryonic development.
Ezrin (clone 21) belongs to a family of proteins known as ERM (ezrin-radisin-moesin) that function as cross-linkers between the actin cytoskeleton and the plasma membrane. Ezrin mRNA and protein are present in the mouse oocyte and in cultured embryos throughout the preimplantation period [29]. The protein molecules are distributed around the cell cortex of blastomeres before compaction, whereas they are restricted to the microvilli of the apical pole of the trophectoderm after compaction [29]. This is consistent with our present result of detecting clone 21 at all embryonic stages in the three developmental conditions studied.
In this study, the expression of clone 21 in in vivo-developed embryos at the 2-cell and 4-cell stage is higher than that in the cultured embryos (P < 0.05). Previous reports and our unpublished data [19, 30] show that in vitro-cultured mouse embryos are associated with more cytoplasmic fragmentation and apoptosis when compared with those developed in vivo. The ERM family members and Rho have been implicated in membrane ruffling and cytokinesis in Swiss 3T3 cells [31]. Ezrin has recently been reported to play a role in determining the survival of cells by activating the phosphatidylinositol-3-kinase/Akt pathway [32]. Whether the high level of ezrin expression would be one of the mechanisms responsible for the better quality of in vivo-developed embryos remains to be investigated.
Clone 41 is homologous to E3KARP, which is a Na+/H+ exchanger isoform 3 (NHE3) kinase A regulatory protein. The NHE3 protein is the epithelial isoform of the Na+/H+ exchanger and is confined to the apical membrane of polarized epithelial cells. It is involved in the absorption of Na+ and HCO3- across the epithelial layer. The NHE3 protein has recently been localized to the apical domain of mouse trophectoderm [33] and is likely to be involved in blastocoel formation.
To the best of our knowledge, the present study is the first to demonstrate the presence of E3KARP in preimplantation embryos. The expression levels of E3KARP are low in early cleaving mouse embryos under the three conditions studied. There is, however, a transient increase in gene expression at the morula stage; E3KARP binds to an internal region within the NHE3 C-terminal cytoplasmic tail and to the carboxyl-terminal domain of ezrin [34]. Evidence suggests that the activity of NHE3 is regulated by the actin cytoskeleton [35], and that E3KARP and possibly ezrin as well are necessary for the cAMP-mediated inhibition of NHE3 by allowing NHE3 to be phosphorylated [36, 37]. The concentration of cAMP in preimplantation mouse embryos increases at the blastocyst stage [38] and cAMP stimulates the rate of blastocoel expansion [39] by stimulating sodium uptake [40]. Thus, it is possible that the transient increase in clone 41 at the morula stage of in vivo-developed mouse embryos (Fig. 3) may prevent premature blastocoel expansion as the presence of E3KARP allows cAMP-mediated inhibition of NHE3. This inhibition is, however, decreased at the blastocyst stage when the expression of E3KARP is low.
In conclusion, the results of this study suggest that the DDRT-PCR-based strategy is a reliable technique for identifying differentially expressed genes in mouse embryos. We showed that the DD profiles of in vivo-developed, in vitro-cultured, and cocultured mouse embryos were essentially similar with some unique bands present in the different culture groups. These differentially expressed DD amplicons can be used as markers to dissect the molecular changes of mouse embryos during preimplantation development.
ACKNOWLEDGMENTS
We thank the clinical staff in the Department of Obstetrics and Gynaecology, The University of Hong Kong for supplying the human oviductal samples, K.L. Kwok for technical assistance, and C.Y.L. Lee and S.K. Chan for embryo culture and collection.
FOOTNOTES
First decision: 26 September 2000.
1 Supported by a grant from the Research Grant Council of the Hong Kong University Grant Committee (HKU7333/97M) to W.S.B.Y. 
2 Correspondence: Kai-Fai Lee, Department of Obstetrics and Gynaecology, Queen Mary Hospital, The University of Hong Kong, Pokfulam Rd., Hong Kong, China. FAX: 852 2855 0947; ckflee{at}hkucc.hku.hk
Accepted: November 2, 2000.
Received: July 31, 2000.
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