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Female Reproductive Tract |
a Department of Obstetrics and Gynaecology
and
b Department of Surgery, Queen Mary Hospital, The University of Hong Kong, Hong Kong, China SAR
c Department of Zoology, The University of Hong Kong, Hong Kong, China SAR
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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early development, embryo, fallopian tubes, gene regulation, oviduct
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and fibroblast growth factor [6–8]. Granulocyte-macrophage colony-stimulating factor, with a peak expression during the preimplantation period in the human fallopian tube [9], also improves human embryo development in a sequential culture system [10]. In addition to growth factors, embryotrophic factors (ETFs) with unknown identities have also been reported [3, 11–14]. However, detection of the embryotrophic activity of these factors is difficult, because the actions of oviductal ETFs are only facilitatory to embryonic development. That is, the embryos can grow in vitro without embryotrophic factors, though their growth is much enhanced after coculture with oviductal cells. Thus, in coculture experiments using mouse embryos, the sensitivity of detecting oviductal-derived embryotrophic activity is often low, because the development of many mouse strains does not require additional support in standard culture condition for blastulation, the usual end point in these studies. This "ETF-independent growth" of the embryo creates high background noise in the detection of embryotrophic activity in coculture. To increase the sensitivity of detection, we used a mouse strain with low developmental potential in commonly used mouse culture media (Chatot-Ziomek-Bavister [CZB] and KSOM) as an assay/model to assess the coculture embryotrophic effect. The normality of preimplantation development of the mouse in this model is reflected by the ability of the embryos to implant after culture and coculture [14]. With this model, we detected the presence of three embryotrophic fractions in spent medium derived from human oviductal cell culture. These fractions, designated as ETF-1, ETF-2, and ETF-3 [15], accumulate in the culture medium with duration of culture [16]. They are heat and protease labile, and they have a differential temporal effect on mouse preimplantation embryo development in vitro [14, 15]. They are unlikely to be related to commonly known growth factors because of their large molecular sizes (>100 kDa).
The identities of the ETFs are not yet known because of the difficulty in isolating sufficient amounts of these fractions from conditioned medium of primary oviductal cells that have only a limited proliferative life span in culture. To solve this problem, we recently established an immortalized human oviductal cell line (OE-E6/E7) that retains most of the characteristics of primary oviductal cells, including the production of oviduct-specific glycoprotein and a glycoprotein fraction with chromatographic behavior similar to that of ETF-3 from primary oviductal cells [17]. To facilitate large-scale affinity purification of ETF-3 for identification purposes, monoclonal antibodies against ETF-3 have been raised. The characterization of these antibodies is ongoing.
Among the three ETFs isolated, ETF-3 is most abundant [14]. It enhances development of the trophectoderm cells of mouse embryos to blastocysts having a larger diameter and a higher hatching rate [3]. This is in line with our previous observation that human oviductal coculture enhances the hatching rate of human embryos in vitro [18], suggesting that ETF-3 may be active in both human and mouse embryos. The detailed mechanism of action of ETF-3 is unknown. We recently demonstrated that coculture alters embryonic mRNA expression using mRNA differential display reverse transcription-polymerase chain reaction (DDRT-PCR) [19]. The DDRT-PCR is a sensitive procedure allowing direct comparisons of the entire mRNA pools between different embryo samples [20]. By employing short random primers in the 5' end in coordination with an anchored oligo-dT primer, RT-PCR can be carried out and differentially expressed cDNA easily isolated with use of a modest number of embryos. In the present study, we have provided evidence that OE-E6/E7 produced ETF-3 and have compared the mRNA expression profile of blastocysts with or without ETF-3 treatment using DDRT-PCR.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The conditioned media derived from primary human oviductal cells and immortalized oviductal cells, OE-E6/E7, were fractionated as previously described [14]. Briefly, the cells were grown in Dulbecco modified Eagle medium/Ham F12 (DMEM/F12) supplemented with 0.3% (w/v) BSA (Sigma, St. Louis, MO). Fifty milliliters of oviductal cell conditioned medium were passed through a concanavalin A column using a fast-performance liquid chromatographic system (Amersham Pharmacia Biotech, Uppsala, Sweden). The column was washed with a start buffer (20 mM Tris [pH 7.4], 0.5 M NaCl, 1 mM MgCl2, 1 mM CaCl2, and 1 mM MnCl2) at a flow rate of 0.3 ml/min for 30 min to remove unbound molecules. The bound glycoproteins were eluted with the same buffer containing 0.3 M -D-methylglucoside at a flow rate of 0.3 ml/min, concentrated by ultrafiltration through the Centricon-100 (Amicon, Inc., Beverly, CA), and further fractionated by a Mono-Q column using the SMART System (Amersham Pharmacia Biotech). The ETF-3 was eluted from the column with 20 mM Tris-HCl (pH 7.5) containing 0.3 M NaCl at a flow rate 70 µl/min. The purified fraction was desalted, concentrated by the Centricon-100, and reconstituted with the appropriate medium.
Embryo Culture
The protocol for obtaining mouse embryos was approved by the Committee on the Use of Live Animal for Teaching and Research, the University of Hong Kong. Mature MF1 female mice (age, 6–8 wk) were superovulated with 5 IU of eCG (Sigma), followed by an injection of 5 IU of hCG (Sigma) 46 h later. The MF1 female mice were then mated with proven-fertile BALB/c males. The day with the presence of vaginal plug was regarded as Day 1. The zygotes were recovered 24 h post-hCG from the oviductal ampullae into Hepes-buffered CZB (CZB/HEPES) [21] containing 0.8 mg/ml of hyaluronidase (Sigma) to remove the cumulus mass. They were washed three times in 250 µl of CZB/HEPES, followed by one wash in CZB, before being pooled and allocated randomly in groups of 20–30 for culturing in CZB with or without ETF-3 supplementation for the first 48 h. They were then transferred to CZB containing 5 mM glucose (CZB+G) for culture. The ETF-3 was supplemented to CZB+G for embryos in the following 72 h.
The percentages of embryos reaching fully expanded blastocyst and hatching blastocyst were recorded at approximately 120 and 144 h post-hCG, respectively. In vivo-developed mouse blastocysts were also collected by flushing the mouse uteri at 104 h post-hCG. The image of each expanded blastocyst was captured with a phase-contrast inverted microscope. The diameter of the expanded blastocyst was determined using the MetaMorph imaging system (version 3.51; Universal Imaging Corp., West Chester, PA) and compared between the ETF-3-treated and medium-alone culture groups. The data obtained from at least three replicate experiments were combined and analyzed by chi-square test or Student t-test when appropriate. Blastocysts were stored at -70°C after image capture for subsequent mRNA expression studies.
Production of Anti-ETF-3 Antibody
Two male BALB/c mice were immunized with 100 µg of ETF-3 in 200 µl of emulsion containing equal volume of PBS and complete Freund adjuvant (Sigma) by i.p. injection (Day 0). Booster injections were similarly given on Days 21 and 35 with 100 µg of ETF-3. Antibody titers against ETF-3 in serum or cultured supernatant were measured by ELISA.
Effect of Anti-ETF-3 Antibody on Embryo Development
To determine whether the antiserum could neutralize the embryotrophic effect of ETF-3, the antiserum was added at different concentrations (3%, 0.3%, and 0.03% [v/v]) to CZB medium supplemented with 10 µg/ml of ETF-3. The same volume of normal mouse serum was added to CZB as control. The ETF-3-supplemented media with or without anti-ETF-3 antibody treatment were used to culture mouse embryos as described above. The development of the embryos at the blastocyst and hatching-blastocyst stages was determined.
Immunostaining of ETF-3 in Cultured Oviductal Cells
Primary human oviductal cells, OE-E6/E7, fibroblasts, and Vero cells (African Green Monkey kidney cell line) were seeded in chamber slide (Nunc, Inc., Naperville, IL) and cultured with serum supplemented with DMEM/F12. Two days later, the cell layers were rinsed with PBS, fixed with 4% (v/v) paraformaldehyde in PBS (pH 7.35) for 30 min, permeated with 0.1% (v/v) Triton X-100 on ice for 2 min, and rinsed six times with PBS. The cells were then blocked in 3% (w/v) BSA/PBS overnight and incubated with anti-ETF-3 antibody (1:2000 [v/v] in 0.3% BSA/PBS containing 0.1% Tween-20) for 2 h, followed by six washes with PBS. The fluorescein isothiocyanate (FITC)-labeled rabbit anti-mouse IgG antibody (1:1000 [v/v] in 0.3% BSA/PBS containing 0.1% Tween-20; Sigma) was then incubated with the cells in a humidified incubator at 37°C for 1 h. Fluorescent staining was observed with a fluorescent microscope after washing off the excess secondary antibody.
Blastocysts with or without ETF-3 treatment were fixed in 4% formaldehyde in PBS (pH 7.35) for at least 30 min and preserved in the same medium for no longer than 1 wk. The embryos were then washed six times in PBS containing 0.3% (w/v) polyvinylpyrrolidone (PVP/PBS), permeated in 0.1% Triton X-100 on ice for 1 min, washed three times in PVP/PBS, and incubated in 3% (w/v) BSA/PBS overnight. After three washes in PVP/PBS, the blastocysts were incubated with anti-ETF-3 antiserum (1:2000 [v/v]) for 2 h. The embryos were then washed six times in PVP/PBS and incubated with FITC-labeled rabbit anti-mouse IgG antibody (1:1000 [v/v] in 0.3% BSA/PBS and 0.1% Tween-20) for 1 h. Finally, the embryos were washed and observed under a fluorescent inverted microscope.
Messenger RNA Isolation from Mouse Blastocysts
Messenger RNAs from eight mouse blastocysts were extracted by Dynabeads mRNA Direct Kit (Dynal AS, Oslo, Norway) as previously described [19]. Messenger RNAs were eluted with 44 µl of diethyl pyrocarbonate-treated water and were used directly for cDNA synthesis.
Differential Display Reverse Transcription-Polymerase Chain Reaction
The RT procedure was performed according to the First Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech). Messenger RNAs from the mouse blastocysts were 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 then incubated at 37°C for 1 h, followed by incubation at 95°C for 5 min. The PCRs were done using the Differential Display Kit (Display Systems Biotech, Vista, CA) as previously described [19]. Briefly, 2 µl of first-strand cDNA were used for amplification. The PCRs were carried out in 1x PCR buffer (10 mM Tris-HCl [pH 7.3], 50 mM KCl, and 1.5 mM MgCl2), 2 µM dNTP, 0.5 µM arbitrary decamer, 2.5 µM anchor primer, 0.1 µl of [-33P]dATP (3000 Ci/mmol; Dupont NEN, Boston, MA), and 0.5 U of Taq DNA polymerase (Roche Molecular Biochemicals, Indianapolis, IN). The PCR was carried out at 94°C for 30 sec, 40°C for 90 sec, and 72°C for 60 sec for 40 cycles in a Perkin-Elmer model 480 thermocycler (Perkin-Elmer Applied Biosystems, Foster City, CA). The reaction was ended with incubation at 72°C for 5 min. The amplified products were separated in a 6% denaturing polyacrylamide gel. The gel was dried, and autoradiography was carried out using Kodak BioMax MR film (Eastman Kodak, Rochester, NY).
Extraction and Reamplification of DD Amplicons
Selected differentially expressed bands were excised from the gel, transferred to centrifuge tubes, and purified as previously described [19]. The DD amplicons were cloned into pGEM-T easy vector (Promega Corp., Madison, WI) and sequenced by an ABI 310 genetics 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 submitted to a BLAST analysis for identification [22].
Semiquantitative RT-PCR Assay
Semiquantitative RT-PCR was used to determine the relative changes of mRNA transcripts in mouse blastocysts that developed in vivo and were cultured in CZB alone and CZB supplemented with ETF-3 from OE-E6/E7 cells. Gene-specific primers for the differentially expressed mRNAs were designed (Table 1). One tube Access RT-PCR was followed according to the manufacturer's protocol (Promega) and as previously described [19]. Briefly, mRNAs from eight blastocysts collected from each group were isolated using Dynabeads mRNA Direct Kit (Dynal AS) as mentioned above. Reverse transcription was first performed at 48°C for 45 min, followed by inactivation of the avian myeloblastosis virus reverse transcriptase at 94°C for 2 min. The PCR immediately followed, and the conditions were 94°C for 30 sec, 60°C for 1 min, and 68°C for 2 min for 26–38 cycles. A constant amount of exogenous human ß-globin RNA was added to each PCR reaction tube for normalization of mRNA amplification. The ß-globin was synthesized using the Maxiscript T7 Transcription Kit (Ambion, Inc., Austin, TX) and quantified by ultraviolet spectrophotometry. A 2.5% NuSieve 3:1 (w/w) agarose (FMC BioProducts, Rockland, MN) was used to resolve the PCR products. The images were captured and the relative amount of amplified products quantified by Labwork Image Acquisition and Analysis Software (UVP, Inc., Upland, CA). A minimum of four replicates of semiquantitative RT-PCR were performed using different batches of mouse blastocysts.
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Statistical Analysis
Data regarding mRNA expression were analyzed using the SigmaStat software package (Jandel Scientific, San Rafael, CA). One-way ANOVA (followed by multiple pairwise comparisons using the Student-Newman-Keuls method) was used for analyzing the difference of mRNA expression assayed by semiquantitative RT-PCR. Differences of P <0.05 were considered to be significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The effects of ETF-3 on embryo development with or without anti-ETF-3 antiserum are shown in Table 2. Treatment with ETF-3 significantly enhanced the blastulation rate (67% vs. 33%) and the hatching rate (45% vs. 16%) of mouse embryos compared to the untreated control. Embryos cultured in the presence of 0.03–3% antiserum in the absence of ETF-3 had similar blastulation and hatching rates as the controls. However, addition of antiserum at the same concentrations abolished the embryotrophic effect of ETF-3. On the other hand, normal mouse serum at concentrations of 0.03–3% had no effect on embryo development.
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ETF-3 Immunoreactivity in Cultured Cells and Blastocysts
With the use of anti-ETF-3 antiserum, immunoreactivity was detected in both the primary human oviductal and OE-E6/E7 cells but not in the Vero cells and human fibroblasts (Fig. 1). These data suggested that the production of ETF-3 was cell-type specific. The ETF-3 immunoreactivity was also detected in the blastocysts treated with ETF-3 derived from the immortalized oviductal cells. Blastocysts from medium-alone culture did not possess the immunoreactivity (Fig. 2).
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Messenger RNA DD
A combination of 10 arbitrary decamer primers and 4 two-base anchored oligo-dT primers (dT11VV, where V = A, C, or G) were used to analyze the mRNA expression profile in mouse blastocysts developed in different culture conditions. Messenger RNAs from mouse blastocysts developed in vivo or cultured in CZB alone or in CZB supplemented with ETF-3 derived from primary or immortalized oviductal cells were used as templates for the DDRT-PCR.
In the present study, we confined our analysis to one developmental stage, expanded blastocyst, which was defined as those with blastocoel occupying more than 50% of the size of the blastocyst and showing zona thinning at 120 h post-hCG. This definition was applied to both the ETF-3-treated and the control blastocysts. Treatment with ETF-treatment was known to produce blastocysts with a larger diameter. Therefore, an increase in the mean diameter of the blastocysts was used in the present study to indicate that the batch of blastocysts had responded to ETF-3 treatment. After confirmation that the batch of blastocysts had an increased diameter, all expanded blastocysts were randomly collected in groups of eight blastocysts for mRNA extraction. Representative diagrams of mouse blastocysts in different treatment groups are shown in Figure 3. The sizes of the expanded blastocysts after ETF-3 treatment, as determined by the diameter of the expanded blastocysts, were significantly larger than those of the control blastocysts (primary oviductal cells, 105 ± 1.17 µm; OE-E6/E7, 101 ± 1.27 µm; control, 93 ± 2.14 µm; P < 0.0001).
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Among the mRNA expression profiles, 34 amplicons produced by 10 arbitrary decamer primer and 4 two-base anchored oligo-dT primers (dT11VV, where V = A, C, or G) clearly showed differential expressions. A representative diagram of the DDRT-PCR pattern is shown in Figure 4. Transcripts of mouse blastocyst that were differentially expressed in different culture conditions were excised. Of 31 reamplified transcripts, 12 were cloned, sequenced, and analyzed. The expression pattern and identities of these transcripts are summarized in Table 3. Of the 12 amplicons, 3 of them (clones 13, 22, and 25) were mouse cDNA clones of unknown functions. Clones 12 and 29 were ribosomal protein S4 (X-linked) and RNA-binding motif protein (X chromosome retrogene), respectively. Clones 1, 2, 5, 11, 14, 20, and 21 showed high sequence homology with mouse ezrin (99%, X60671), heat shock 70-kDa protein (HSP70; 98%, NM_022310), cytochrome c oxidase subunit VIIa-L precursor (COXVIIIaL; 100%, AF037371), proteinase-activated receptor 2 (PAR-2; 91%, Z48043), eukaryotic translation initiation factor 2ß (eIF-2ß; 90%, BC003848), cullin 1 (Cul-1; 98%, AF136441), and proliferating cell nuclear antigen (PCNA; 98%, BC005778), respectively (Table 3).
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Analysis of Blastocyst mRNA by Semiquantitative RT-PCR
To confirm differential expression of the above 12 mRNAs, gene-specific primers were designed and used for one-step RT-PCR. The primer sequences and the cycles used for the amplification are listed in Table 1. Messenger RNAs were extracted from mouse blastocysts developed in vivo or cultured either in CZB alone or in ETF-3-supplemented CZB. Four to five different batches of mouse blastocyst mRNA were subjected to RT-PCR. A representative diagram of mRNA transcript expression level in different mouse blastocysts is shown in Figure 5. The relative amount of amplified products of the 12 mRNAs in blastocysts of different treatment groups from RT-PCR match well with the patterns in DDRT-PCR. Expression of clones 1, 2, 11, 12, 14, 20, and 29 was higher in both in vivo-developed and ETF-3-treated blastocysts than in vitro-cultured blastocysts. The expression of clone 13 was higher in the ETF-3-treated blastocysts compared to those in the other two groups. Clones 5, 21, 22, and 25 had higher expression in the in vivo-developed blastocysts only.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The DDRT-PCR is a well-established method [20] that has been used to study mRNA expression in embryos [19, 23, 24]. In the present study, the DDRT-PCR banding patterns of embryos treated with ETF-3 derived from primary cells and OE-E6/E7 are more similar to each other than to the embryos cultured in medium alone or developed in vivo. Twelve differentially expressed mRNAs were isolated for confirmation by RT-PCR. Seven of them (clones 1, 2, 11, 12, 14, 20, and 29) have higher expression in both in vivo-developed blastocysts and in blastocysts treated with ETF-3 derived from OE-E6/E7 or primary oviductal cells (data not shown) by semiquantitative RT-PCR. Five of the seven genes (PAR-2, Cul-1, ezrin, HSP70, and eIF-2ß) have been implicated in embryogenesis. The remaining two (ribosomal protein S4 [X-linked] and RNA-binding motif protein) have not been documented to be involved in development of the preimplantation embryo.
Expression of PAR-2 protein is first detected at mouse Embryonic Day 12 and persists throughout embryogenesis [25]. To the best of our knowledge, the present study is the first demonstrating the presence of PAR-2 mRNA in mouse blastocysts. The PAR-2 mRNA is highly expressed in the in vivo-developed and the ETF-3-treated blastocysts compared with those cultured in medium alone (Fig. 5). The PAR-2 is a member of the family of protease-activated, G protein-coupled receptors. The proteinase-activated receptors are activated enzymatically, through proteolysis of the receptor, rather than through simple ligand occupancy [26]. In keratinocytes, activation of PAR-2 leads to the release of interleukin-6 (IL-6) [27]. Both IL-6 and its receptor (IL-6R) are present in human blastocyst [28], and functional IL-6 is also secreted from mouse blastocyst [29]. Interestingly, transient expression of IL-6 mRNA was suggested to be involved in the formation of a capillary network in the maternal decidua during early postimplantation development [30]. It is possible that ETF-3 may activate PAR-2 of the treated embryos, resulting in the release of IL-6 that, in turn, acts on the uterus to facilitate implantation or on the blastocysts as an autocrine agent. Recently, Nance and Priess [31] showed that proteinase-activated receptors were involved in the cell polarity, gastrulation, and blastocoel formation in Caenorhabditis elegans embryos and that PAR-2 was localized to the basal and lateral surfaces of the embryonic cells. The exact function of PAR-2 in mammalian embryo development warrants further investigation.
Clone 20 was found to be Cul-1. The Cul-1 protein is part of the Skp-cullin-F-box complex, which regulates the abundance of cell-cycling proteins at the G1-S phase [32]. Implanted Cul-1 mutant (Cul-1-/-) mouse embryos fail to develop beyond Embryonic Day 5.5 and are almost completely resorbed by 7.5 days [33], indicating the importance of Cul-1 in embryogenesis. In culture, the abnormal development of Cul-1-/- embryos is detectable at around Embryonic Days 5.5–6.5 and is associated with increased expression of p53 and apoptosis in the embryonic ectoderm [33] and with accumulation of cyclin E [34]. How Cul-1 deficiency affects development is unclear. Recent data showed that c-Myc promoted G1 exit of cell cycling in part via Cul-1-dependent ubiquitination and that enforced expression of Cul-1 was capable of overcoming the slow-growth phenotype of c-Myc-deficient mouse embryonic fibroblasts [35]. These data are consistent with our observation that higher expression of Cul-1 was associated with better development of in vivo-developed blastocysts and ETF-3-treated blastocysts when compared with blastocysts cultured in medium alone. In this connection, ETF-3-treated embryos have a higher blastomere count per blastocyst than those derived from culture in medium alone [3].
The mRNA expression of ezrin in mouse embryos increased with development [19]. Its mRNA and protein are present in mouse embryos throughout preimplantation development [36]. Ezrin is a member of ezrin-radixin-moesin family [37], which links the actin cytoskeleton to the plasma membrane and is involved in microvilli formation and cell adhesion. The cellular location of ezrin changes during compaction; it is distributed around the cell cortex of blastomere before compaction but is localized to the microvilli of the apical pole of the trophectoderm after compaction [36]. The higher mRNA expression of ezrin after ETF-3 treatment may be one of the factors leading to better and larger blastocysts compared with the control, because ezrin has been implicated in the control of ionic pump activity [38] and cavitation of the blastocyst [39].
In mouse embryos, HSP70 is detectable at the 2-cell stage and has the highest expression at the blastocyst stage [40]. Heat shock proteins are stress proteins involved in facilitating protein folding and assembly and in stabilizing the damaged protein for repair and degradation in cells [41], and they may protect the embryo from undergoing apoptosis [42]. Culture of embryos with antibody against HSP70 is associated with more DNA fragmentation and growth inhibition of the treated embryos [42, 43]. Endometrial cell coculture reduces these detrimental effects of anti-HSP antibody [44]. Human oviductal cells also reduce the incidence of apoptosis in cocultured embryos [1]. The results of the present study are consistent with these observations. The higher expression of HSP70 in ETF-3-treated and in vivo-developed blastocysts may indicate that these embryos are more tolerant of developmental stress when compared with blastocysts cultured in medium alone.
Translation is a critical point for the regulation of gene expression during embryo development [45]. Eukaryotic translation initiation factor (eIF) 1A is transiently increased during embryonic genome activation in mouse and bovine embryo with or without oviductal cell coculture [19, 46]. In the present study, the expression of eIF-2ß (clone 14) was higher after ETF-3 treatment than without treatment. Eukaryotic translation initiation factor 2 is a heterotrimer consisting of three subunits (, ß, and 
). It forms a complex with GTP and promotes the binding of methionyl-tRNA to ribosomes during the initiation of protein translation [47]. The expression of eIF-2 is higher in mouse blastocyst than in other preimplantation stages [46], and its regulatory functions in protein translation have been well documented [47, 48]. However, to our knowledge, no previous report has been published regarding the expression of eIF-2ß in mouse blastocysts. The implication of the change in eIF-2ß after ETF-3 treatment is unclear.
Clones 21 and 5 were highly expressed in the in vivo-developed embryos only as determined by RT-PCR. They are PCNA and COXVIIaL, respectively. The PCNA is a specific marker of the cell-cycle S phase [49] and an important component of the DNA replication and repair machinery [50]. The higher expression of PCNA in the in vivo-developed blastocysts may indicate better quality of these embryos compared with those in the other two groups. The COXVIIaL is the liver-type isoform of COXVIIa, which is a nuclear-coded subunit in the cytochrome c oxidase family [51]. Cytochrome c oxidase is the terminal protein complex of the electron-transport chain. How its expression relates to embryonic growth is unclear, but COXVIIaL is the major type of COXVII isoform found during fetal development [52].
In conclusion, both primary and immortalized oviductal cells synthesize and secrete ETF-3. Although development of the mouse embryo in vitro is suboptimal, the present study shows ETF-3 treatment can maintain the expression of certain mRNAs in the treated blastocysts to a level comparable with those in embryos developed in vivo. Apart from having a predominant effect on trophectoderm development, ETF-3 also has some stimulatory effect on development of the inner cell mass after a 4-day treatment [15]. The latter could be caused by the general growth-promoting effect of ETF-3 on embryos. Which of these ETF-3 effects (or if both) contribute to the differential mRNA expression in this study is unknown. The present study is a first step in examining the mechanism of action of ETF-3, and it identifies a number of potential ETF-3 target genes. Future experiments will concentrate on the effect of ETF-3 on the mRNA and protein expression of these genes in cells of the trophectoderm and inner cell mass. An understanding of the mechanisms of action of ETF-3 would allow us to design better medium for embryo culture. The immortalized oviductal cells would allow production of sufficient amount of ETF-3 for identification and further characterization of this ETF.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: W.S.B. Yeung, Department of Obstetrics and Gynaecology, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, China SAR. FAX: 852 2855 0947; wsbyeung{at}hkucc.hku.hk
Received: 5 June 2002.
First decision: 30 June 2002.
Accepted: 12 August 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ß receptors and contain immunoreactive GM-CSF protein. J Clin Endocrinol Metab 1994 79:662-665[Abstract] by protein kinase GCN2 mediates gene-specific translational control of GCN4 in yeast. Cell 1992 68:585-596[CrossRef][Medline]This article has been cited by other articles:
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  A. W.Y. Cheong, Y.-L. Lee, W.-M. Liu, W. S.B. Yeung, and K.-F. Lee Oviductal Microsomal Epoxide Hydrolase (EPHX1) Reduces Reactive Oxygen Species (ROS) Level and Enhances Preimplantation Mouse Embryo Development Biol Reprod, July 1, 2009; 81(1): 126 - 132. [Abstract] [Full Text] [PDF]
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 P.-K. Tse, Y.-L. Lee, W.-N. Chow, J. M. C. Luk, K.-F. Lee, and W. S. B. Yeung Preimplantation Embryos Cooperate with Oviductal Cells to Produce Embryotrophic Inactivated Complement-3b Endocrinology, March 1, 2008; 149(3): 1268 - 1276. [Abstract] [Full Text] [PDF]
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K.-F. Lee, J.-S. Xu, Y.-L. Lee, and W. S. B. Yeung Demilune Cell and Parotid Protein from Murine Oviductal Epithelium Stimulates Preimplantation Embryo Development Endocrinology, January 1, 2006; 147(1): 79 - 87. [Abstract] [Full Text] [PDF]
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 J.S. Xu, Y.L. Lee, K.F. Lee, K.L. Kwok, W.M. Lee, J.M. Luk, and W.S.B. Yeung Embryotrophic factor-3 from human oviductal cells enhances proliferation, suppresses apoptosis and stimulates the expression of the {beta}1 subunit of sodium-potassium ATPase in mouse embryos Hum. Reprod., December 1, 2004; 19(12): 2919 - 2926. [Abstract] [Full Text] [PDF]
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 Y.-L. Lee, K.-F. Lee, J.-S. Xu, Q.-Y. He, J.-F. Chiu, W. M. Lee, J. M. Luk, and W. S. B. Yeung The Embryotrophic Activity of Oviductal Cell-derived Complement C3b and iC3b, a Novel Function of Complement Protein in Reproduction J. Biol. Chem., March 26, 2004; 279(13): 12763 - 12768. [Abstract] [Full Text] [PDF]
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