Mol. Hum. Reprod. Advance Access originally published online on February 4, 2005
Molecular Human Reproduction 2005 11(3):173-181; doi:10.1093/molehr/gah155
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Oct-4 mRNA and protein expression during human preimplantation development
1Research Centre Genetics and Reproduction, University Hospital and Medical School, Brussels Free University (Vrije Universiteit Brussel), Laarbeeklaan 101, 1090 Brussels, Belgium
2 To whom correspondence should be addressed. Email: gcauffma{at}az.vub.ac.be
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Abstract |
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The transcription factor OCT-4 is regarded as a critical factor in controlling mammalian early embryonic development because of its role in toti-/pluripotency. In human preimplantation embryos, OCT-4 studies are limited to RNA analysis of abnormally developing embryos. This study thoroughly investigated the expression pattern of OCT-4 throughout the human preimplantation development. Expression was examined by single-cell RT–PCR or indirect immunocytochemistry in 36 single oocytes of various maturity and 112 normally developing preimplantation embryos at the level of single blastomeres, morulas, blastocysts, or inner cell mass (ICM) and trophectoderm (TE) samples. Oocytes and cleavage stage embryos revealed a variable OCT-4 expression pattern, concomitant with a pure cytoplasmic localization of the protein. During compaction, the variability in expression faded away indicating embryonic OCT-4 expression and the protein appeared in the nucleus implying biological activity. In blastocysts, OCT-4 transcripts and proteins were present in the ICM and the TE. At protein level, blastocysts displayed different spatial expression patterns within a cell for the splice variants of OCT-4, which may endow them with different functional properties. As OCT-4 transcripts were also found in various differentiated cells, the presence of OCT-4 transcripts or proteins may not be sufficient for identifying undifferentiated cell lines in humans. Further, we suggest to examine the localization of OCT-4 proteins within a cell rather than to look for the presence and/or amount of transcripts.
Key words: expression/human embryos/OCT-4/pluripotent/preimplantation development
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Introduction |
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One of the POU domain related transcription factors is regarded as a key factor allowing mammalian cells to remain in the cycle of totipotency (Pesce et al., 1999
). This transcription factor is variously known as OCT-4, OCT-3, OCT-3/4, POU5f1, OTF3, or NF-A3. Confusion is already generated by the naming of Oct-4 and also more vagueness exists about the conservation of its expression pattern among all mammals.
In mice, Oct-4's association with the toti-/pluripotent cell type has been established (Yeom et al., 1996; Ovitt and Schöler, 1998; Pesce et al., 1998; Pesce and Schöler, 2001; Pan et al., 2002). During the murine preimplantation period, a residual maternal Oct-4 expression is found in unfertilized oocytes and in the subsequent cleavage stages (Palmieri et al., 1994). Embryonic Oct-4 expression is initiated at the 4–8-cell stage and is concomitant with an abundant expression in the nuclei of all blastomeres. Upon blastocyst formation, Oct-4 is down-regulated in the trophectoderm (TE) and becomes restricted to the inner cell mass (ICM) (Palmieri et al., 1994; Mitalipov et al., 2003) where it is required for the maintenance of the pluripotent character (Nichols et al., 1998; Kim et al., 2002). In murine embryonic stem cells (Rosner et al., 1990; Tanaka et al., 2002), Oct-4 controls cell fate in a dose-dependent way (Niwa et al., 2000). Embryonic germ (Yeom et al., 1996) and embryonic carcinoma cell lines (Lenardo et al., 1989; Okamoto et al., 1990) also show an abundant expression of Oct-4, in which differentiation is associated with a progressive loss of Oct-4 expression. Since the murine Oct-4 orthologue genes, including human, bovine and porcine, are highly conserved, a similar role has been suggested for Oct-4 in all mammals.
In humans, OCT-4 is the product of the OTF3 gene. Unlike in mice, two splice variants, designated OCT-3A and OCT-3B, are encoded (Takeda et al., 1992). Although expression of OCT-3A and OCT-3B has not yet been fully investigated, the OCT-4 expression studies are usually carried out without considering the existence of both splice variants, and thus without considering possible differences in their expression. In addition, OTF3-related genes, such as the retroposon OTF3C have also been described (Takeda et al., 1992). OCT-4 is found to be expressed throughout all stages from the unfertilized oocyte to the blastocyst, as detected by PCR amplification of cDNA libraries (Verlinsky et al., 1998) and by RT–PCR (Abdel-Rahman et al., 1995; Hansis et al., 2000, 2001; Huntriss et al., 2002). In blastocysts, the expression of OCT-4 was observed in the ICM and the TE (Hansis et al., 2000; Huntriss et al., 2004), but titration studies revealed a higher level of expression in the ICM (Hansis et al., 2000). As in mice, the expression of OCT-4 has been established in human embryonic stem (hES) cells (Reubinoff et al., 2000), human embryonic carcinoma cells (Pera and Herszfeld, 1998) and human embryonic germ cells (Goto et al., 1999; Turnpenny et al., 2003), and upon differentiation, the OCT-4 expression was lost. However, human non-embryonic carcinomas, such as breast, pancreas and colon carcinomas (Monk and Holding, 2001; Wang et al., 2003), early-differentiated hES cells (Assady et al., 2001; Henderson et al., 2002; Richards et al., 2003; Schulz et al., 2003), fetal testis (Clark et al., 2004), amniotic fluid cells (Prusa et al., 2003), and adult tissues and cells, such as heart, liver, spleen, kidney, placenta, pancreatic islets (Takeda et al., 1992), and cultured fibroblasts (Verlinsky et al., 1998), express OCT-4 as well.
In the present study, the expression pattern of OCT-4 was thoroughly investigated throughout the human preimplantation development. Using RT–PCR, the expression was assessed in normally developing preimplantation embryos and also in single oocytes and in various differentiated cell types. Embryos prior to compaction were examined at the level of single blastomeres, whereas compacted embryos and blastocysts were examined at the level of single embryos. To investigate a possible difference in OCT-4 expression between the pluripotent and the differentiated embryonic cells, some blastocysts were split into ICM and TE samples. Using indirect immunocytochemistry, the OCT-4 protein expression was examined for the first time in human oocytes and preimplantation embryos. Both splice variants, OCT-3A and OCT-3B were considered.
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Samples
With the approval of the institutional ethical committee and the couple's informed consent, oocytes, preimplantation embryos, cumulus cells, semen and testicular tissues were obtained at our Centre for Reproductive Medicine. Female patients underwent controlled ovarian stimulation (Kolibianakis et al., 2004
) and oocyte retrieval (Platteau et al., 2003). Oocytes were denuded from surrounding cumulus and corona cells by using a combination of enzymatic (40 IU/ml hyaluronidase type VIII; Sigma Aldrich, Belgium) and mechanical (pipetting) methods (Van de Velde et al., 1997). Some cumulus cells were collected for analysis. The oocytes used were immature, at either the germinal vesicle (GV) stage or the metaphase I stage (MI), or were in vitro matured until the metaphase II stage (MII), or were fresh MIIs donated for research. Embryos from the 2-cell stage until the blastocyst stage were obtained after conventional IVF or ICSI (Staessen et al., 2003; Devroey and Van Steirteghem, 2004) and assessed as unsuitable for transfer or freezing. Some embryos were obtained by applying ICSI on in vitro matured or fresh MII oocytes donated for research. The embryos were cultured in 25 µl of G1/G2 medium (Vitrolife, Sweden) in an incubator (37°C, 5% O2, 5% CO2, 90% N2) (Heraeus; Meyvis, Belgium). Embryo cleavage was evaluated daily on the basis of number, size, cell shape and fragmentation rate of the blastomeres (Staessen et al., 2003). Morulas were scored according to their degree of compaction, whereas blastocyst evaluation relied on the scoring system described by Gardner and Schoolcraft (1999). Briefly, a first score was given to the blastocyst depending on its stage of expansion, ranging from early over full, expanded, and hatching to hatched. From the stage of full blastocyst onwards, a second and third score were given to the ICM and the TE, respectively: ranging from A (tightly packed, many cells), over B (loosely grouped, several cells) to C (very few cells) for the ICM and from A (many cells forming a cohesive epithelium) to B (few cells forming a loose epithelium) for the TE. An additional D (degenerative) score was introduced for both ICM and TE. Only embryos derived from normally fertilized oocytes (2PN) with a normal morphology and normal developmental timing were used. Semen and testicular tissues were obtained from the male partners of couples at the infertility centre. Ejaculated sperm samples had normal semen parameters (WHO, 1999). Fresh testicular tissues were obtained from patients who underwent vasectomy repair and who showed normal spermatogenesis afterwards. Cultured human skin fibroblasts and lymphoblasts were obtained from the Centre for Medical Genetics.
RT–PCR
Sample preparation
Individual oocytes and embryos were placed in droplets of acidic Tyrode's (AT) solution of pH 2.4 (14 mM NaCl, 0.2 mM KCl, 0.2 mM CaCl2.2H2O, 0.05 mM MgCl2.6H2O, 5.5 mM glucose) by using a mouth-controlled hand-drawn Pasteur pipette and observed under a stereomicroscope (40x magnification, Wild, Leica; Van Hopplynus, Belgium) until the zona pellucida (ZP) had just dissolved. Single oocytes and embryos were transferred to droplets of in-house prepared Ca2+- and Mg2+-free medium [14 mM NaCl, 0.2 mM KCL, 0.04 mM NaH2PO4.H2O, 5.5 mM glucose, 1.2 mM NaHCO3, 0.02 mM EDTA, 0.01% (w/v) phenol red] supplemented with 4 mg/ml bovine serum albumin (BSA) (Sigma Aldrich) and washed three times. Embryos prior to full compaction were dissociated into single blastomeres by repeatedly in- and outpipetting. Washing was repeated for each blastomere. Some expanded blastocysts with clearly distinguishable ICMs were split into one sample containing only TE cells (TE sample) and another sample containing the ICM with its surrounding TE cells (ICM sample). These blastocysts of which the ZP was not removed were transferred to pre-incubated droplets of HEPES-buffered Earle's injection medium and stabilized with a micromanipulator on an inverted microscope (400x magnification; Nikon Narishige, Japan). Using a non-contact 1.48 µm diode laser system (Fertilase; Octax, Germany), a hole was created in the ZP at the level of the ICM. The ICM was pulled out of the ZP using a biopsy pipette. Subsequently, the ICM sample was separated from the blastocyst by laser. Great care was taken to avoid any ICM cell contamination in the TE sample by leaving some space between the ICM cells and the cut. Both samples were moved to different parts of the droplet and the ZP was separated from the TE sample by in- and outpipetting. Since, initially, the ZP was not removed, only ICSI blastocysts were used in order to avoid contamination by surrounding spermatozoa and cumulus cells. Little clumps of cumulus cells (
10 cells) were washed three times in Ca2+- and Mg2+-free medium. Media samples were consistently taken to serve as negative controls. The individual oocytes, blastomeres, compacted embryos, blastocysts, ICM and TE samples, and clumps of cumulus cells were directly transferred to RT-tubes containing 2.5 µl of ice-chilled lysis buffer [0.8% Igepal® (Sigma Aldrich), 1 U RNaseOUTTM (Invitrogen, Belgium), 5 mM dithiothreitol (Sigma Aldrich)] made in diethyl pyrocarbonate (DEPC)-treated H2O (Daniels et al., 1997). Samples were immediately snap-frozen in liquid nitrogen for 30 min to avoid endogenous RNase activity and were then used in RT reactions.
Sperm samples were pooled after purification on a two-layer Pure Sperm 90–45% density gradient (Nicadon International AB, Sweden) and washed with in-house prepared HEPES-buffered Earle's medium. Testicular tissues were transferred to cryo-tubes and stored in liquid nitrogen until use. Lymphoblasts were cultured according to standard procedures (Ventura et al., 1988). Colonies were collected and washed three times in 500 µl phosphate-buffered saline (PBS). One to five million cells were isolated for RNA extraction. Cultured fibroblasts were trypsinized and centrifuged for 10 min at 300 g. The pellet was washed two times in 0.9% NaCl. The dry pellet was used for RNA extraction. Total RNA extraction from sperm samples, testicular tissues, lymphoblasts and fibroblasts was performed using the Spin Protocol of the RNeasy Mini kit (Qiagen, The Netherlands) according to the instructions of the manufacturer. Testicular tissues were first ground with a pestle. RNA concentration was determined by spectrophotometry (GeneQuant II; Amersham Pharmacia Biotech Inc., The Netherlands). All samples were stored at –80°C until use.
RT
RT was performed using the First-strand cDNA Synthesis Kit (Amersham Pharmacia Biotech Inc.) with the NotI-d(T)18 primer. For sperm samples, testicular tissues, lymphoblasts and fibroblasts, RT was carried out with 1–5 µg of total RNA in a 33 µl reaction according to the instructions of the manufacturer. For oocytes, blastomeres, compacted embryos, blastocysts, ICM and TE samples, and clumps of cumulus cells, the manufacturer's protocol was adapted for single cells: samples were incubated at 80°C for 5 min to disrupt the secondary and tertiary structures in the RNA and were immediately transferred to ice. The RT mix (RT reagents for a 15 µl reaction supplemented with 2 U RNaseOUTTM dissolved in DEPC-treated H2O) was subsequently added to the samples. RT was carried out at 37°C for 1 h in an Eppendorf Mastercycler Personal (VWR International, Belgium). Samples were immediately returned to ice.
Prior to PCR, the completed first-strand reaction was heated to 90°C for 5 min to denature the RNA–cDNA duplexes and inactivate the reverse transcriptase. Finally, the preparation was chilled on ice.
PCR
PCR for OCT-4 was carried out by using intron-spanning primers consisting of the forward primer (5'-GACAACAATGAGAACCTTCAGGAGA-3') labelled with 5' indocarbocyanine (Cy5) and the reverse primer (5'-TTCTGGCGCCGGTTACAGAACCA-3') (Eurogentec, Belgium) (Abdel-Rahman et al., 1995). These primers amplified a fragment of 218 bp that could be addressed to both OCT-4 splice variants. Four microlitres of cDNA was used as the template in a 25 µl final reaction volume comprising 1x PCR buffer (Applied Biosystems, The Netherlands), 10 pmol of each primer and 1.25 U AmpliTaq DNA Polymerase (Applied Biosystems). PCR was carried out in an Eppendorf Mastercycler Personal using the following cycling profile: 5 min denaturation at 95°C followed by 50 cycles of 30 s at 95°C, 30 s at 55°C and 30 s at 72°C, and a final extension for 7 min at 72°C. cDNA amplifications of sperm samples, testicular tissues, cumulus cells, fibroblasts and lymphoblasts were carried out as described above, except that 2 µl of template were used instead of 4 µl and that 30 cycles were run instead of 50 cycles. Co-amplification of the HPRT cDNA sequence was carried out for each sample and served as a positive control for cell viability as well as for the efficacy of the RT procedure. The HPRT primers were also intron-spanning and PCR conditions were identical to those of OCT-4. The Cy5-labelled forward primer (5'-GCCGGCTCCGTTATGGCG-3') and the reverse primer (5'-AGCCCCCCTTGAGCACACAGA-3') (Eurogentec) amplified a fragment of 226 bp (Ao et al., 1994). To sort out amplification failure from non-expression, a positive control of OCT-4 and HPRT amplification on cDNA from lymphoblasts was run with each series of samples. The negative controls, taken during sample preparation, were included in each experiment and processed in the same way as the samples. All PCR fragments were analysed on an ALF-express automated sequencer (Amersham Pharmacia Biotech Inc.). Results were processed using the Allelelinks software provided by the manufacturer.
OCT-4 RT–PCR products from lymphoblasts and testicular tissue were verified by sequencing analysis on the ABI 310 (Applied Biosystems).
Indirect immunocytochemistry
Immunocytochemistry was carried out with either an affinity-purified goat polyclonal antibody against OCT-4 (C-2O: sc-8629; Santa Cruz Biotechnology, Tebu-bio, Belgium) whose epitope maps at the carboxy terminus of OCT-4 and therefore recognizes both OCT-4 splice variants (OCT-3A/B) or a mouse monoclonal IgG2b antibody against OCT-4 (C-1O: sc-5279; Santa Cruz Biotechnology, Tebu-bio) whose epitope corresponds to amino acids 1–134 of OCT-4 and therefore only recognizes OCT-3A. Prior to both staining procedures, oocytes and embryos were exposed briefly to AT solution for zona thinning or complete zona removal and washed three times in PBS supplemented with 1% BSA. Staining with the C-20 antibody was done as follows. Oocytes and embryos were fixed in PBS containing 3.7% formaldehyde (Merck, VWR International) for 30 min at room temperature. The samples were subsequently washed and permeabilised by incubation for 1 h at room temperature in a detergent solution containing 0.1% Triton X-100 (Sigma Aldrich) and 0.1% Igepal® in PBS. After washing, oocytes and embryos were incubated overnight with the C-20 antibody at a concentration of 2 µg/ml at 4°C. Samples were washed and incubated with a 1:200 dilution of a rabbit anti-goat fluorescein isothiocyanate (FITC)-conjugated secondary antibody (F9012; Sigma Aldrich) for 2 h at 4°C in the dark and then washed again. The primary and secondary antibody solutions were prepared in PBS supplemented with 2% BSA and 5 µg/ml normal rabbit serum (Sigma Aldrich) in order to avoid non-specific binding of the secondary antibody. The oocytes and blastocysts used for staining with the C-10 antibody were fixed and permeabilised by incubation in cold methanol for 5 min at –20°C. After washing, reaction with the C-10 antibody at a concentration of 10 µg/ml was done overnight at 4°C. Washing was repeated and samples were incubated with a 1:200 dilution of a goat anti-mouse FITC-conjugated F(ab')2 fragment (sc-3699; Santa Cruz Biotechnology, Tebu-bio) for 2 h at 4°C in the dark. Finally, samples were washed. The primary and secondary antibody solutions were prepared in PBS supplemented with 2% BSA.
Control reactions for non-specific immunofluorescence of the anti-OCT-4 antibodies C-20 and C-10 were carried out by replacing them with goat immunoglobulin G (IgG) and with a mouse monoclonal IgG2b antibody against HLA-DR (eBioscience; ImmunoSource, Belgium), respectively, at similar concentrations to those used for the specific primary antibodies. Control reactions for the secondary antibodies were performed by omitting the primary antibodies. In order to control the fixation and permeabilisation procedures, all oocytes and embryos were additionally stained with a direct labelled antibody against actin (Texas Red-X phalloidin, Molecular Probes; VWR International, Belgium) at a concentration of 5 µg/ml prepared in PBS and supplemented with 2% BSA. Reaction took place for 2 h at 4°C in the dark. After final washing, oocytes and embryos were mounted on glass coverslips in Slow Fade Light (Molecular Probes; VWR International) prior to examination or storage at 4°C in the dark. Confocal scanning microscopy with an argon–krypton laser (488/568) (Fluoview IX70; Olympus, Belgium) was used to record the fluorescent images. All images for test antibodies and controls were collected using identical confocal settings and processed identically after collection.
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Results |
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RT–PCR
After adaptation of the RT–PCR technique to the single-cell level, OCT-4 (OCT-3A/B) and HPRT mRNA expression was examined in 18 single oocytes of various maturity, 84 single blastomeres derived from 16 cleavage stage embryos (2–9-cell stage), 13 single blastomeres of a compacting 13-cell embryo, six single compacted embryos and 19 single, intact blastocysts. The results are summarized in Table I. OCT-4 showed a variable expression pattern in oocytes and cleavage stage embryos where transcripts were found in 22.2% (4/18) and 17.3% (14/84) of the examined cells, respectively. In these embryos, mRNA expression varied widely between individual blastomeres from the same embryo and between embryos of the same cell stage (Figure 1). A decline in expression was observed at the late cleavage stages where OCT-4 mRNA was detected in only 6.3% of the blastomeres of 8-cell embryos and in none of the blastomeres of the 9-cell embryo. Later in development, OCT-4 mRNA was found in 84.6% (11/13) of the blastomeres of the compacting embryo. Further, its mRNA was present in all compacted embryos and in all but one blastocyst. HPRT mRNA was detected in all oocytes, 60.7% (51/84) of the blastomeres of cleavage stage embryos, all the cells of the compacting embryo, all compacted embryos, and all but one blastocyst. The negative result in one of the blastocysts for both OCT-4 and HPRT was obtained for the same sample and was probably due to non-viability. The variability in HPRT expression was only found during the third cell division, ranging from 100% at the 4-cell stage to 20% at the 5-cell stage, followed by a gradual increase over 56.3% at the 8-cell stage to 100% at the 9-cell stage.
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Ten expanded blastocysts, graded from AA to DB, were separated into ICM and TE samples. All TE samples and all but one ICM sample showed expression of OCT-4 mRNA. In one blastocyst, HPRT mRNA was absent in the TE sample (Table II, Figure 2).
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Expression of OCT-4 mRNA was also detected in four independent samples of cumulus cells and two independent samples of sperm, testicular tissue, fibroblasts (data not shown) and lymphoblasts (Figure 2, positive control). The authenticity of the OCT-4 RT–PCR products from lymphoblasts and testicular tissue was confirmed by cDNA sequencing (data not shown). Control reactions for HPRT mRNA were positive in all samples with exception of the sperm samples where no HPRT transcripts could be detected.
Transcripts of OCT-4 and HPRT were ubiquitously detected in the PCR control reactions. No amplification of OCT-4 or HPRT was ever achieved from negative controls.
Indirect immunocytochemistry
The intensity of the fluorescent signals varied widely in the samples, ranging from negligible over clear to strong signals. Only clear and strong signals were counted as positive. OCT-4 (OCT-3A/B) protein expression was analysed using the C-20 antibody in 10 oocytes of various maturity, 10 cleavage stage embryos (4–10-cell stage), seven compacted embryos, and 23 blastocysts of various stages and grades. The results are summarized in Table III. Here, variability in expression was also observed between oocytes and between and within cleavage stage embryos from which 70% (7/10) and 63.9% (46/72) of the cells stained positive, respectively. In these cleavage stage embryos, the percentage of OCT-4 positive cells decreased from 100% at the 4-cell stage, over 60% at the 5–8-cell stage, to 11.1% at the 9-cell stage, whereupon it increased over 80% at the 10-cell stage. At later stages, fluorescent signals were observed in all the cells of compacted embryos and blastocysts. The intensity of fluorescence was never higher in the ICM than in the TE, irrespective of the stage of blastocyst expansion. OCT-4 showed a remarkable spacial distribution pattern throughout the developmental stages. In oocytes, the signal was primarily restricted to the cell periphery. From the early cleavage stages till the stage of compaction the OCT-4 protein appeared to be excluded from the nucleus of blastomeres. The compacted embryos showed either a pure cytoplasmic staining, or a diffuse cellular staining, or a mixture of both staining patterns. In blastocysts, the protein was equally distributed between the cytoplasm and the nucleus of the cells. Representative images of staining are shown in Figures 3B–H and 4D–
. Using the C-10 antibody (OCT-3A), a total of eight oocytes of various maturity and 20 blastocysts of various stages and grades were examined (Table III). The observed fluorescent signal was strikingly weaker in comparison with the staining results achieved with the C-20 antibody. The OCT-3A protein showed a weak diffuse cellular distribution pattern in almost all (87.5%) (7/8) oocytes. In all blastocysts, OCT-3A positive signals were observed in both ICM and TE. Seventy per cent of the blastocysts (ranging form early to hatched blastocysts) showed a similar intensity of fluorescence in the ICM and TE (Table IV). The remaining 30% (5 expanded blastocysts and 1 hatching) showed a higher intensity of fluorescence in some cells of the ICM. No relation could be made between the stage of blastocyst expansion and the intensity of the signals in the ICM because from the stage of expansion onwards, as many blastocysts showed an equal intensity of fluorescence between the ICM and the TE as a higher intensity in the ICM. It was notable that the blastocysts showing a brighter staining in their ICM had a better grade for the ICM than for the TE, implying a better quality of the ICM (Table IV). All the cells (ICM and TE) of the blastocysts showed an unequal distribution pattern of the protein, with a high density in the nucleus and a lower density in the cytoplasm. Representative images of staining are shown in Figures 3A and 4A–C.
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The additional staining with the anti-actin antibody was successful in all oocytes and embryos examined. Control reactions for non-specific and background staining gave no fluorescent signals (Figure 3I– P).
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The present study reports the temporal and spatial expression pattern of OCT-4 during early human embryogenesis, from oocyte to blastocyst.
To avoid the generation of misleading expression profiles, only morphologically normal diploid embryos with a normal developmental timing were studied. OCT-4 mRNA analysis was carried out with commonly used primers. The amplified message can be addressed to both OCT-4 splice variants (OCT-3A/B), but not to contamination with genomic DNA, as the primers were designed to amplify an intron-spanning region. Contamination with the pseudogene OTF3C, a retroposon (Takeda et al., 1992), is excluded by sequence analysis and because of the existence of OCT-4 negative cells. Protein expression was investigated using two well-characterized antibodies of which one recognized the OCT-3A and OCT-3B protein and the other only the OCT-3A protein. False positive results due to OTF3C are unlikely, because the amino acid sequence encoded by OTF3C differs from OCT-4 proteins at 15 residues and by the deletion of a triplet corresponding to Gln258.
The results show that OCT-4 mRNA and proteins are expressed throughout the last stages of oogenesis and throughout the whole preimplantation development. Expression was not consistently detected in all cells, as variability was found at particular developmental stages. The phenomenon of variability may be an inherent property of preimplantation embryos reflecting the maternal-to-embryonic transition, which in general take place at the 4–8-cell stage (Tesarik et al., 1986; Braude et al., 1988; Taylor et al., 1997). Unlike for HPRT, OCT-4 does not perfectly fit in this general model. A variable maternal OCT-4 expression is found in oocytes, which are highly differentiated cells, and in the subsequent cleavage stages. In these cleavage stage embryos, variability is seen within an embryo and between embryos of the same cell stage. During the period of compaction, which starts from the 10-cell stage onwards (Nikas et al., 1996), the variability in OCT-4 expression diminishes and OCT-4 becomes consistently expressed in all the cells, indicating embryonic expression. This uncommon expression pattern may imply that the human embryonic OCT-4 expression starts, like in other mammals (Mitalipov et al., 2003, Kurosaka et al., 2004), one to two cell cycles after the general embryonic genome activation. Because Oct-4 operates as a transcription factor, its cytoplasmic localization conjectures that OCT-4 is not involved in biological functions in oocytes and cleavage stage embryos. In compacted embryos, the protein gradually appears in the nucleus, suggesting a functional role. Relating the localization of OCT-4 proteins with the timing of embryonic genome activation, it is most likely that the maternally encoded OCT-4 proteins are not functionally active, whereas proteins encoded by the embryonic genome are. Since OCT-4 proteins can also be found in the cytoplasm, it is of greater value to examine the localization of the OCT-4 proteins within a cell rather than to look for the presence and/or the amount of RNA.
OCT-4 mRNA being present in human unfertilized oocytes and preimplantation embryos confirms earlier reports. However, these reports rely on studies on a small number of oocytes and preimplantation embryos, of which the oocytes were pooled (Huntriss et al., 2002), or possibly contaminated with cumulus cells (Abdel-Rahman et al., 1995; Huntriss et al., 2002) or sperm (Abdel-Rahman et al., 1995), and of which the embryos were mostly abnormally fertilized, not studied at the level of single blastomeres, and also possibly contaminated (Abdel-Rahman et al., 1995; Huntriss et al., 2002). The expression of OCT-4 mRNA in individual blastomeres was previously described (Hansis et al., 2001). Although not all blastomeres of each embryo were examined, a decline was also found in the percentage of OCT-4 positive blastomeres from the 2-cell stage onwards. The study suggested a relation between the higher number of cells in the embryo and the lower number of OCT-4 positive cells and put forward the hypothesis that the variable OCT-4 expression in individual human blastomeres directs cells towards the ICM or TE lineage and hence that OCT-4 could be used as a marker in PGD to identify embryogenic (ICM) blastomeres. However, only embryos up to and including the 10-cell stage were analysed. Our analyses show at RNA level an increase in the number of OCT-4 positive cells in a compacting 13-cell embryo and at the protein level uniform OCT-4 expression in compacted embryos. Given that compaction establishes a polarity of cells within the embryo, which through subsequent cell divisions leads to the formation of two distinct cell lineages (ICM and TE), OCT-4 expression does not direct the cells towards a specific lineage.
Examining a high number of human blastocysts (29 for RNA analysis and 43 for protein determination) revealed that OCT-4 is not restricted to the pluripotent cells of the human preimplantation embryo. OCT-4 mRNA and proteins were present in both cell types of the blastocyst: the ICM, which represents the pluripotent cells of the embryo, and the TE, which is differentiated. Previous reports also demonstrated the expression of OCT-4 mRNA in the ICM and TE (Hansis et al., 2000; Huntriss et al., 2004). Based on titration studies, a 31-fold higher level of OCT-4 mRNA was demonstrated in the ICM, as compared with the TE (Hansis et al., 2000). Although our mRNA study was not quantitative, at the protein level we were able to detect differences in the amount of proteins between the different cells within a blastocyst by comparing the intensity of the fluorescent signals. Investigation of both OCT-4 proteins (OCT-3A/B) on one hand and OCT-3A protein only on the other hand revealed important differences. Detection analysis of OCT-3A/B proteins showed an expression pattern identical to the one found in bovine and porcine blastocysts (van Eijk et al., 1999; Kirchhof et al., 2000). The proteins were equally distributed between the ICM and the TE of all blastocysts and between the cytoplasm and the nucleus of both cell types. Detection analysis of the OCT-3A protein revealed in the majority of the blastocysts similar protein levels between the two cell types. However, the brighter signals in the ICM of some blastocysts cannot be ignored. It is unclear whether this difference is significant because these brighter signals were observed in blastocysts with an inferior TE quality as compared to the excellent ICM quality. Another difference is the unequal intracellular distribution of the OCT-3A protein, as it is mainly localized in the nucleus of ICM and TE cells. Although this intracellular distribution is similar to the one reported in mice and rhesus monkeys, the intercellular distribution is not (Palmieri et al., 1994; Mitalipov et al., 2003). Upon expansion of murine and monkey blastocysts, the signal for OCT-4 diminishes in the TE and at the stage of hatching, OCT-4 expression is completely restricted to the ICM. In our study, the brighter signals for OCT-3A in the ICM were also found from the stage of expansion onwards, but a relation with these stages could not be established.
The possibility of different biological properties of the OCT-3A and OCT-3B protein cannot be ruled out, as our two protein determination studies exposed different spatial expression patterns within a cell. In blastocysts, staining for OCT-3A displayed more intense signals in the nucleus than in the cytoplasm, whereas staining for OCT-3A and OCT-3B together showed an equal cellular distribution of the signal. This might imply that OCT-3A is rather a nuclear protein and OCT-3B is rather a cytoplasmic protein. At the molecular level, OCT-3A and OCT-3B proteins differ in sequence at their NH2-termini. This region of OCT-3A is rich in proline and glycine residues, whereas the NH2-terminus of OCT-3B is not. Since proline-rich regions form part of the transcriptional activation domain of other transcription factors, alternative splicing of OCT-4 mRNA may generate proteins with common DNA-binding domains but different activation domains, thereby endowing OCT-3A and OCT-3B with different functional properties. Sequencing analysis revealed that the mouse otf3 gene encodes a protein similar to OCT-3A but not to OCT-3B. This might imply that OCT-3B does not serve a function that is crucial for mammalian development (Takeda et al., 1992).
Further research on the relation between the OCT-4 expression and the undifferentiated cell type involved RT–PCR of OCT-4 in a number of differentiated cells and tissues. OCT-4 transcripts were detected in all samples. Because of the presence of the transcripts in cumulus cells and spermatozoa, special attention should be paid to the complete removal of these cells when performing RT–PCR on oocytes and embryos, as there is a substantial contamination risk. These findings support previous reported results on OCT-4 expression in human adult tissues (Takeda et al., 1992; Verlinsky et al., 1998). However, in hES cell research, OCT-4 expression analysis, especially at RNA level, has become part of the basic characterization of ES cell lines. OCT-4 transcripts and proteins are found in undifferentiated hES cells but also in early-differentiated hES cells (Henderson et al., 2002; Schulz et al., 2003). Upon further differentiation, OCT-4 mRNA expression is lost (Reubinoff et al., 2000; Zwaka and Thomson, 2003). Artificial down-regulation of OCT-4 expression with RNA interference in hES showed that OCT-4 is required to maintain the undifferentiated stem cell state (Matin et al., 2004). Considering all information about the OCT-4 expression in humans, it seems that all pluripotent cells express OCT-4 and this expression is crucial, but there is no guarantee that when OCT-4 transcripts or proteins are present in a cell, this cell is pluripotent.
In summary, human OCT-4 expression is not restricted to the pluripotent cells of the preimplantation embryo and so, the murine embryonic OCT-4 expression pattern may not be extrapolated to humans. Further, it should be emphasized that in humans there could be a functional difference between the OCT-3A and OCT-3B protein. Additional investigations on the differences in expression between both proteins in different cell types are necessary. We recommend to examine OCT-4's state of biological activity by looking at the localization of the OCT-4 proteins within a cell, rather than looking for the presence and/or amount of OCT-4 transcripts.
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We would like to thank the laboratory staff of the Centre for Reproductive Medicine, especially Isabel Lemahieu for injecting oocytes donated for research and the Follicle Biology Laboratory for putting the confocal microscope at our disposal. We would also like to thank Prof. Dr K. Sermon and Dr M. De Rycke for their support in this project and the latter for scrupulously reading this manuscript. Furthermore, we are grateful to M. Whitburn of the Language Education Centre for proofreading the manuscript. The work was supported by grants from a Concerted Action of the Brussels Free University (Vrije Universiteit Brussel).
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