Mol. Hum. Reprod. Advance Access originally published online on April 2, 2004
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Molecular Human Reproduction, Vol. 10, No. 6, pp. 383-392, 2004
© European Society of Human Reproduction and Embryology 2004
p66shc, but not p53, is involved in early arrest of in vitro-produced bovine embryos
Department of Biomedical Sciences, University of Guelph, Guelph, Ontario, N1G 2W1, Canada
1 To whom correspondence should be addressed. e-mail: waking{at}uoguelph.ca
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ABSTRACT |
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High embryo loss occurs in the first week of bovine embryo development, with a high percentage of embryonic arrest. We hypothesized that arrested embryos enter a ‘senescence-like state’ and that both the cell cycle regulatory protein p53 and the stress-related protein p66shc, which are involved in the onset of senescence in somatic cells, are responsible for this early embryonic arrest. In our in vitro production system, 13.5 ± 0.5% of embryos arrest at the 2–4-cell stage. First cleavage occurs between 26 and 48 h post insemination (hpi), with early cleaving embryos showing only 0.6 ± 0.3% arrest, with later cleaving embryos exhibiting up to 14.2 ± 0.9% arrest. We compared 2–4-cell embryos collected at 28 hpi with those arrested at the 2–4-cell stage collected at day 8 post insemination. Quantification by real-time PCR and by semi-quantitative immunofluorescence showed significantly higher p66shc mRNA and protein levels in both arrested and late cleaving embryos versus 28 hpi embryos. By comparison, no significant changes in p53 mRNA, protein and phosphorylation levels were detected. Taken together, these results demonstrate that embryonic developmental potential is related to the time of first cleavage and that p66shc, but not p53, is up-regulated in early arrested in vitro-produced bovine embryos.
Key words: developmental arrest/embryos/p53/p66shc/senescence
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Introduction |
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In humans, 50% of in vitro-produced (IVP) embryos arrest during the first week of development (Hardy et al., 2001) and only 25% of the embryos that are transferred to patients after IVF implant (Dawson et al., 1995). In cattle, fewer than half of IVP embryos reach the blastocyst stage (Xu et al., 1992) and many of these do not implant or attach following embryo transfer (Betts and King, 2001).
The reasons for this high rate of early developmental failure remain unclear. Cells with the characteristic features of apoptosis, such as cytoplasmic, nuclear and DNA fragmentation, have been detected in both arrested and developing embryos, suggesting that apoptosis, an essential mechanism for the removal of unwanted cells during normal development (Hardy, 1999), might also play a role in embryonic arrest (Hardy, 1997; Kamjoo et al., 2002). In mouse embryos, early developmental arrest has been associated with elevated levels of free oxygen radicals, indicating that oxidative stress might be the reason for such a high incidence of arrest (Johnson and Nasr-Esfahani, 1994). Early embryo arrest has been proposed as a protective mechanism for preventing further development of abnormal embryos, as 47.9% of arrested human embryos showed chromosomal abnormalities (Almeida and Bolton, 1998). Moreover, abnormalities are more frequent not only in arrested embryos, but also in embryos with slow or accelerated cleavage, emphasizing the importance of the time of first cleavage for correct development (Yadav et al., 1993; Magli et al., 1998, 2001). A clear relationship between the time of first cleavage and developmental potential has been previously demonstrated for both bovine and human oocytes, with the earliest cleaving oocytes being more likely to develop to the blastocyst stage than those that cleave late (Plante et al., 1994; Lonergan et al., 1999, 2000; Ward et al., 2001; Fenwick et al., 2002; Racowsky, 2002; Lequarre et al., 2003). It was also observed that timing of first cleavage is associated with different stability of specific transcripts necessary for correct development (Brevini et al., 2002) and differential maternal mRNA expression (Fair et al., 2004). The time of first cleavage can also be affected by the sex of the embryo as well as by different culture environments (Yadav et al., 1993; Peippo et al., 2001).
Development through the early cleavage stages, before the activation of the embryonic genome occurring at 8–16 cells in the bovine (Nothias et al., 1995; Schultz, 2002), is mainly controlled by stored maternal factors (Memili et al., 1998; Wu et al., 2003). However, recent studies reported that some embryonic transcription is active from the zygote stage in the bovine species (Plante et al., 1994; Memili and First, 2000) and that less than optimal in vitro conditions cause arrest before genome activation (Bavister, 1995; Rizos et al., 2002). Furthermore, the arrest that occurs at these early stages of development might be related to apoptosis or to embryos entering a senescence-like state.
Senescence is the in vitro phenomenon, observed first by Hayflick, whereby somatic cells stop dividing in vitro after a fixed number of population doublings (Hayflick and Moorhead, 1961; Sozou and Kirkwood, 2001). The onset of senescence is associated with the shortening of telomeres, highly conserved DNA sequences of (TTAGGG)n tandem repeats at the end of chromosomes (Olovnikov, 1973; Harley et al., 1990; Wright et al., 1996) and other morphological characteristics. A mechanism for telomere length maintenance providing chromosomal stability throughout gametogenesis and early embryogenesis has been previously shown in bovine oocytes and early embryos (Betts and King, 2001). However, recent studies suggest a more centralized role of the tumour suppressor protein p53 rather than telomere length in the induction of cellular senescence in somatic cells (Sharpless and DePinho, 2002).
The tumour suppressor protein p53 is critical for embryonic response to environmental stresses and regulates differentiation during embryo development (Lichnovsky et al., 1998). p53 eliminates embryonic stem cells damaged by teratogens through a p53-dependent apoptosis and embryos lacking p53 progress through the cell cycle despite the presence of DNA damage, resulting in an increased incidence of developmental abnormalities (Stewart and Pietenpol, 2001). There are different opinions on the relevance of p53 during embryonic development. For instance, Jurisicova et al. (1998) detected high p53 levels in the inner cell mass of mouse blastocysts, suggesting that p53 is necessary for apoptosis to occur. Conversely, Frenkel et al. (1999) suggest that apoptosis could occur in the mouse embryo, in either a p53-dependent or -independent way; however, the rate at which apoptosis proceeded in the absence of p53 was found to be considerably slower. In bovine preimplantation embryos, a lack of p53 nuclear localization was detected, suggesting that p53 might not be active during early bovine embryo development (Matwee et al., 2000). Together, these observations suggest that the relative importance and/or function of p53 during early embryonic development have not been fully elucidated yet.
We previously demonstrated that during cellular senescence p53 was up-regulated in fetal bovine fibroblasts and was also associated with an increase in the expression of the recently identified stress-related protein p66shc (L.A.Favetta et al., unpublished data). p66shc has been associated with differences in lifespan in mice and propagation of apoptogenic signals in mouse fibroblasts (Luzi et al., 2000; Ventura et al., 2002). The lifespan of p66shc–/– mice was 30% longer than wild type mice and, interestingly, the p53 apoptotic response to UV radiation and hydrogen peroxide treatment in cell cultures derived from these knockout mice was reduced (Migliaccio et al., 1999; Lithgow and Andersen, 2000). Together these observations suggest that p66shc is associated with apoptosis and, more importantly, may also be associated with the induction of cellular senescence.
Here we investigated the morphological and biochemical features of arrested embryos to correlate the percentage of arrest with the time of first cleavage and we measured p53 and p66shc expression in relation to early arrest and time of first cleavage. At the early developmental stages, embryos use stored maternal transcripts (Latham and Schultz, 2001), therefore proteins that result over-expressed in arrested embryos might be used as screening molecules to assess oocyte competence and to identify those embryos that will arrest.
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Materials and methods |
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In vitro bovine embryo production
Bovine ovaries were obtained from a local abattoir (J.M.Schneider, Inc., Canada). Cumulus–oocyte complexes (COC) were collected by follicular aspiration and a modified version of the in vitro oocyte maturation, fertilization, and embryo culture methods described by Betts et al. (1997) was employed for the production of bovine preimplantation embryos. Briefly, follicular contents were aspirated and the COC were collected into HEPES-buffered Ham’s F-10 (Sigma–Aldrich Canada, Canada), supplemented with 2% steer serum. The COC were washed twice in maturation medium consisting of TCM-199 (Sigma–Aldrich) supplemented with 2% bovine serum (Cansera), 0.2 mol/l sodium pyruvate (Sigma–Aldrich), 0.2 mol/l L-glutamine (Sigma–Aldrich), 0.6% penicillin–streptomycin (Invitrogen, Canada) and once in maturation medium with the addition of 0.1 µg/ml of FSH, 1 µg/ml of LH and 1 µg/ml of estradiol (NIH, USA). COC were matured in 80 µl drops of maturation medium with hormonal supplement under silicon oil (Paisley Products, Canada) for 22 h at 38.5°C in a humidified atmosphere of 5% CO2 in air. Matured oocytes were washed three times in 500 µl of HEPES-buffered Tyrode’s albumin–lactate–pyruvate medium (HEPES/Sperm TALP), as per Parrish et al. (1988) protocol. Approximately 30 COC were placed into each 100 µl drop of fertilization medium, IVF–TALP [HEPES/Sperm TALP, supplemented with 20 µg/ml heparin (Sigma–Aldrich)] under silicone oil (Paisley Products). Frozen–thawed bovine semen (Gencor, Canada) was prepared by swim-up in HEPES/Sperm TALP medium for 1 h at 38.5°C in a humidified atmosphere of 5% CO2 in air before centrifugation at 300 g for 10 min. COC were co-incubated with sperm at a final concentration of
1x106 motile sperm/ml for 16–18 h at 38.5°C in a humidified atmosphere of 5% CO2 in air. Following co-incubation, the remaining cumulus cells were removed by gentle vortexing. Presumptive zygotes were transferred to 50 µl drops of in vitro culture (IVC) medium, consisting of TCM-199 supplemented with 0.2 mol/l sodium pyruvate, 0.6% penicillin–streptomycin, and 0.1% polyvinylalcohol (PVA) (Sigma–Aldrich) under oil. Groups of 25–30 presumptive zygotes were co-cultured with 20 bovine oviductal epithelial cell vesicles for up to 9 days. To sustain development through to the blastocyst stage, 20 µl of fresh IVC medium was added to each drop after 48 h of culture.
Evaluation of cleavage, blastocyst and arrest rates
The cleavage rate was evaluated at day 2 post insemination, whereas blastocyst development was evaluated at day 8–9 post insemination with embryos considered ‘arrested’ when appearing as 2–4-cell embryos at day 8 post insemination.
For the experiment investigating p53 and p66shc gene expression patterns at different developmental stages, GV oocytes were collected after aspiration from ovaries, metaphase II (MII) oocytes were collected after 22 h of maturation, 2–4-cell embryos were collected up to 40 hpi, 8-cell embryos were collected at 60–72 hpi, morulae were collected at day 6 post insemination and blastocysts were collected at day 8 post insemination. After collection, embryos were washed in phosphate-buffered saline (PBS) supplemented with 0.1% PVA (PBS/PVA), snap-frozen in liquid nitrogen and then stored at –80°C until RNA was extracted. Embryos were collected in pools of 100 at the germinal vesicle (GV), MII and 2–4-cell stage, in pools of 50 at the 8-cell and morula stage and in pools of 30 at the blastocyst stage. Data were normalized per one oocyte/embryo. GV and MII oocytes were removed of their surrounding cumulus cells by vortexing before freezing.
For the experiment relating percentage of embryo arrest and time of cleavage, embryos were separated in different IVC drops at 26, 28, 30 and 32 hpi. Percentage of cleavage was evaluated at the corresponding time of cleavage analysed, percentage of blastocyst development was evaluated at day 8–9 post insemination and calculated as number of blastocysts divided by number of embryos cleaved at the specific hpi for that experiment and percentage of arrested embryos was calculated at day 8 post insemination.
For the experiment investigating gene expression at 28 hpi versus day 8 arrested embryos and 28 hpi versus late cleaving embryos, embryos were collected at 28 hpi (early cleaving embryos) and between 29 and 48 hpi (late cleaving embryos). Arrested embryos were collected at day 8 post insemination. Pools of 100 embryos were snap-frozen in liquid nitrogen and stored at –80°C until RNA was extracted or embryos were fixed in PBS containing 4% paraformaldehyde (PFA) (Fisher Scientific, Canada) and stored in the refrigerator for histological examination.
TUNEL assay
Embryos were collected at the desired stage and washed three times in PBS/PVA and then fixed with 4% PFA in PBS for 1 h at room temperature. Fixed embryos were permeabilized in 0.5% Triton X-100 (Biorad Laboratories Ltd, Canada) in PBS for 1 h at room temperature and processed for DNA fragmentation, using an adaptation of the TUNEL protocol described by Byrne et al. (1999) (In situ detection kit; Roche Molecular Biochemicals, Canada). Briefly, embryos were incubated in the TUNEL mixture (deoxynucleotidyl transferase enzyme and fluorescein-dUTP in a 1:9 ratio) for 1 h at 37°C in the dark. Positive control embryos were preincubated with 5 IU DNase I (Promega, USA) for 30 min at 37°C. As a negative control, embryos were incubated in fluorescein-dUTP in the absence of the enzyme. Embryos were then washed twice in 0.5% Triton in PBS, once in PBS/PVA and once in RNase buffer (40 mmol/l Tris, 10 mmol/l NaCl, 6 mmol/l MgCl2, pH 8) before incubation with 0.1 mg/ml of RNase (Sigma–Aldrich) for 1 h at 37°C in the dark. Following two washes in RNase buffer, the nuclei were stained with propidium iodide (PI) (5 µg/ml) for 45 min at 37°C in the dark. Finally, embryos were mounted on slides with Dako® fluorescent mounting medium (Dako Corp., USA) after a quick wash in PBS/PVA and analysed by fluorescence microscopy (Aristoplan; Leitz). Only the nuclei that showed fragmented and condensed morphology by PI staining and positive for TUNEL staining were considered apoptotic.
Total RNA extraction
RNA was extracted from each pool of 30–100 embryos using QIA ShredderTM and RNeasy® Mini Kit (Qiagen Inc., Canada), according to the manufacturer’s instructions. The RNA was co-precipitated using 2 µl of SeeDNA (Amersham Biosciences Corp., Canada) as a carrier by the addition of 0.1 volume of 3 mol/l sodium acetate (pH 5.2) (Amersham Biosciences Corp.) and 2 volumes of ice-cold absolute ethanol. The RNA pellet was air-dried and resuspended in 8 µl of RNase-free water. Samples then underwent DNase treatment (DNA-freeTM; Ambion Inc., USA). To each sample, 1 µl of DNase I and 1 µl of 10xDNase buffer were added and incubated at 37°C for 30 min. To inactivate the enzyme, 5 µl of DNase Inactivation Reagent was added and after a 2 min incubation at room temperature, the samples were centrifuged at 10 000 g. The supernatant was then collected.
Reverse transcription
RT reactions were performed on the RNA extracted from pools of oocytes/embryos using 500 ng of oligo (dT)12–18 and Superscript II (Invitrogen) reverse transcriptase. The oligo (dT)12–18 was first added to the RNA samples and allowed to anneal by denaturing the secondary structures at 70°C for 2 min, then a mixture of 4 µl RT buffer, 1 µl of dithiothreitol 0.1 mol/l, 1 µl of 10 mmol/l dNTP mix, 0.5 µl of RNAsin (40 IU/µl) (Promega) and 1 µl of Superscript II (200 IU/µl) was added and the reaction was incubated at 42°C for 1 h, followed by a denaturing step at 70°C for 30 min.
Primers
To amplify the cDNA of interest we used the p53 primers previously described by Matwee et al. (2000), p66shc primers previously used in fetal bovine fibroblasts (L.A.Favetta et al., unpublished data) and primers for H2a described by Robert et al. (2002). The sequences and information on the source of the primers are listed in Table I. To confirm the specificity of the primers, the three gene products were amplified using liver RT reactions, which were performed using standard procedure on 5 µg of total RNA. A cDNA aliquot of 1 µl was subjected to PCR amplification in the GeneAmp PCR System 2400 Perkin Elmer Thermocycler. To each sample the following mixture was added: PCR buffer 10x, 1.5–3 mmol/l MgCl2, 2.5 mmol/l dNTP mix, 100 pmol of each forward and reverse primer, 2.5 IU Ampli Taq GoldTM polymerase (Applied Biosystems) and water to a final volume of 50 µl. Each product underwent the following program of amplification: activation at 95°C for 10 min and then 35–40 amplification cycles of denaturation at 95°C for 1 min, annealing for 1 min, and elongation at 72°C for 1 min. The cycles were followed by a final elongation step at 72°C for 10 min. Aliquots of the PCR products were separated on a 2% agarose gel; the specific band (Table I) for each gene was extracted from the gel using Qiaquick gel extraction columns (Qiagen Inc.). The purified PCR products were quantified by running an aliquot on a 2% agarose gel along with a low DNA mass ladder (Invitrogen), and 20 ng of each product at a concentration of 10 ng/µl was sent to be sequenced (University of Guelph Molecular Supercentre) to confirm their identity and used to calibrate the real-time PCR assay.
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Real-time PCR
Purified PCR products were quantified by running an aliquot along with low DNA mass ladder (Invitrogen) and diluted for the real-time PCR standard curves. The PCR were conducted in a Light Cycler apparatus (Roche Molecular Biochemicals) and products were detected with SYBR Green (FastStart Master SYBR Green I mix; Roche Molecular Biochemicals). Prior to the quantification, optimization procedures were performed by running PCR with or without the purified template to identify the melting temperatures of the primer dimers and the specific product. To measure the level of mRNA in the samples, the fluorescence values were taken at the temperature associated with the beginning of the peak for the specific product (Table I). For each quantification, a 1 µl aliquot of the RT reaction was used. The standard curve was established using the DNA prepared as described above and six serial dilutions were used ranging from 100 pg to 1x10–4 pg. The amplification program was as follows: preincubation for FastStart polymerase activation at 95°C for 10 min, followed by 40 amplification cycles of denaturation at 95°C (20°C/s), annealing for 5 s (20°C/s), elongation at 72°C and acquisition of fluorescence (Table I) for 5 s (20°C/s). After the end of the last cycle, the melting curve was generated by starting the fluorescence acquisition at 72°C and taking measurements every 0.1°C until 95°C was reached. Amplification was performed on H2a gene as an endogenous standard to assure equal RT efficiency.
Semi-quantitative immunofluorescence
Embryos were washed twice in PBS/PVA and incubated for 3–5 min with 0.1% pronase (pre-warmed at 37°C) to eliminate the zona pellucida and rinsed twice in PBS/PVA. Embryos were then fixed with 4% PFA in PBS for 1 h at room temperature and then washed three times in PBS/PVA before permeabilization in 0.5% Triton X in PBS for 1 h at room temperature. Embryos were then washed twice in PBS/PVA and incubated overnight with the primary antibody diluted in 3% bovine serum albumin (BSA) (Sigma–Aldrich) in TBS at 4°C. Optimization experiments for each antibody were performed separately to ensure the use of a concentration detectable, but not saturating the substrate. For both the p53 (Cell Signalling Technology® Inc., USA) and serine 20 phospho p53 antibodies (Cell Signalling Technology® Inc.) the optimal dilution was 1:100 in 3% BSA in TBS, whereas the optimal dilution for the p66shc antibody (Migliaccio et al., 1999) was 1:250 in 3% BSA in TBS. Embryos were washed twice for 5 min in 0.5% Triton in PBS and then in PBS/PVA prior to incubation with a fluorescein isothiocyanate-conjugated goat anti-rabbit secondary antibody (Sigma–Aldrich) diluted 1:80 in 3% BSA in TBS. After two washes for 5 min in 0.5% Triton in PBS, embryos were incubated with 0.1 mg/ml of RNase in RNase buffer for 1 h at 37°C in the dark. Following two washes in RNase buffer, the nuclei were stained with PI (5 µg/ml) for 45 min at 37°C in the dark and rinsed in PBS/PVA. Finally embryos were mounted on slides with Dako® fluorescent mounting medium (Dako Corp.). Images were acquired and the number of pixels was measured using the software metamorph (Meta Imaging, series 5.0) connected to a fluorescence microscope (Olympus BX61).
Statistical analysis
Statistical analysis was performed using one-way ANOVA, when comparing three or more groups and two-sample t-test, when comparing two groups. The non-parametric equivalent tests were used when samples did not meet the assumption of normal distribution or homogeneity of variance. Data were analysed by MinitabTM and means were considered significantly different when P < 0.05.
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Results |
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Assessment of early embryo arrest
In our IVP system, 13.5 ± 0.5% of the total number of fertilized oocytes and 15.6 ± 0.6% of cleaved embryos arrest at the 2–4-cell stage (Table II). As seen by light microscopy, 2–4-cell arrested embryos appear intact with a morphology consistent with the 2–4-cell stage up until day 8 post insemination, when the surrounding embryos already reached the blastocyst stage (Figure 1).
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Examination of apoptosis in early embryo arrest
To ensure that the arrested embryos were not dying through apoptosis, we investigated the presence of DNA fragmentation in 28 hpi 2-cell embryos compared to 2–4-cell arrested embryos collected at day 8 post insemination using TUNEL analysis. We were not able to detect DNA fragmentation by TUNEL staining in either group (Figure 2C, D). It has been previously shown that very early stages of development (zygote to 4-cell stage) do not appear to undergo apoptosis (Byrne et al., 1999; Matwee et al., 2000) and specifically Gjørret et al. (2003) observed a lack of DNA fragmentation by PI staining at the 2-cell stage in bovine IVP embryos. In the latter study, the earliest detected signs of DNA fragmentation appeared at the 3–8-cell stage. In our experiments, we were able to detect three fragmented nuclei by PI staining in 28 hpi embryos, but no TUNEL staining in those nuclei. Analogous results were observed in 2–4-cell arrested embryos collected at day 8 post insemination, as summarized in Table III. These data allow us to conclude that apoptosis does not occur in either 28 hpi embryos or in 2–4-cell arrested embryos.
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Quantification of p53 and p66shc during in vitro bovine embryo development
Maternal mRNA accumulates during oogenesis and oocyte maturation ensuring protein synthesis during genome silencing (Paynton and Bachvarova, 1994) and is gradually degraded until the resumption of embryonic transcription latter in development. This explains the sharp increase in RNA levels at the morula/blastocyst stages. Most genes follow this profile of expression during development, for example the many genes known as ‘housekeeping genes’, like tubulin, GAPDH, ubiquitin and lamin B (Robert et al., 2002), retinoblastoma (Iwamori et al., 2002),
and ß subunits of the Na,K-ATPase pump (Watson et al., 1990; Betts et al., 1997) and apoptosis-related genes (Jurisicova et al., 2003). Our results show that the p53 mRNA expression profile follows the analogous pattern as previously described, with high mRNA levels at the oocyte stage, low expression at the early stages of development and increasing expression at the morula and blastocyst stages, after the activation of the embryonic genome (Figure 3A). Interestingly, the p66shc mRNA expression profile is quite different from what was expected, displaying a gradual decrease from the GV oocyte stage to the blastocyst stage (Figure 3B). The expression profile of H2a mRNA was used as an endogenous control to assure equal RT efficiency, as it has been shown to remain constant during different stages of development (Robert et al., 2002) and the p53 and p66shc data were normalized towards it.
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Evaluation of embryo arrest and blastocyst development in relation to time of cleavage
To determine an optimal control group for the comparison of the 2–4-cell arrested embryos in the subsequent experiments, we quantified the percentage of 2–4-cell arrest in groups of embryos cleaving at different hpi. As shown in Figure 4A, embryos cleaving up to 28 hpi show a significantly lower (P < 0.001) percentage of 2–4-cell arrest than embryos cleaving later (32 hpi). Percentage of blastocyst development in each group was also reported as an index of developmental ability (Figure 4B). Interestingly, very early cleaving embryos (26 hpi) show significantly lower blastocyst development (9.1 ± 0.6%), suggesting that although very early cleaving embryos do not result in a high percentage of 2–4-cell arrest (0.6 ± 0.6%), they are less capable of fully developing to the blastocyst stage. On the other hand, later cleaving embryos (32 hpi) are more likely to arrest (14.2 ± 0.9%), but only 26.5 ± 1.7% of them are able to fully develop to blastocyst, versus >40% blastocyst rate in the 28 and 30 hpi cleaving embryos (P < 0.001). These results suggest that embryos cleaving at 28 hpi, with decreased percentage of arrest (0.6 ± 0.3%) and increased blastocyst development (41.2 ± 2.1%), are the embryos that have the greatest potential for producing viable offspring.
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p66shc and p53 mRNA expression in early arrested embryos
No differences in p53 mRNA levels (P = 0.83) were detected between our control group (28 hpi) and 2–4-cell embryos arrested and collected at day 8 post insemination (Figure 5A). In contrast, p66shc mRNA levels were significantly increased in arrested embryos compared to 28 hpi cleaving embryos (P < 0.0001) (Figure 5B). Quantification of H2a mRNA levels in the two groups was used as a control, and the p53 and p66shc data were normalized towards it. These results suggest that p66shc might play a role in early embryo arrest.
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p66shc and p53 mRNA expression in relation to time of first cleavage
Since our results showed that late cleaving embryos are more likely to undergo early embryo arrest, we quantified the expression of p53 and p66shc mRNA between early cleaving embryos (28 hpi) and later cleaving embryos (between 29 hpi and 48 hpi) to investigate whether over-expression of p66shc was already present at the 2 cell stage. This would further support p66shc as a possible initiator of early embryo arrest. No changes in p53 mRNA expression were detected between early and late cleaving embryos (P = 0.377) (Figure 6A), but significantly higher (P < 0.01) expression of p66shc mRNA was detected in late cleaving embryos, those more likely to undergo arrest, compared to 28 hpi cleaving embryos (Figure 6B). These data strengthen the possible role of p66shc in inducing early embryo arrest. Quantification of H2a mRNA levels in the two groups was used as an endogenous control, and the p53 and p66shc data were normalized to it.
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Protein expression of p66shc and p53 and serine 20 p53 phosphorylation levels in late cleaving embryos versus early cleaving embryos
To ascertain whether the results obtained at the mRNA level were reflected in the different abundance of their respective proteins, we quantified p53 and p66shc protein abundance in 28 hpi cleaving embryos and later cleaving embryos, between 29 and 48 hpi, using semi-quantitative immunofluorescence. No differences (P = 1.00) in p53 abundance were detected in the two groups of embryos examined (Figure 7B–D), whereas a significantly higher (P < 0.001) amount of p66shc was observed in late cleaving embryos (Figure 7J–D). In somatic cells we observed that the onset of senescence might not only involve the abundance of p53, but also p53 phosphorylation levels. In particular, we found p53 to be over-phosphorylated in serine 20 at the onset of senescence (L.A.Favetta et al., unpublished data). Therefore, this led us to investigate serine 20 p53 phosphorylation levels between the two groups of early and late cleaving embryos. No differences (P = 0.689) in serine 20 p53 phosphorylation levels were detected in early and late cleaving embryos (Figure 7F–H), suggesting that p53 is not involved in the early arrest of IVP bovine embryos.
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Discussion |
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TopABSTRACTIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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The aim of this study was to gain a better understanding of embryo arrest at the early cleavage stages of in vitro development, before the major activation of the embryonic genome. We investigated the morphological characteristics of embryos that remained at 2–4 cells up until day-8 post insemination. Evidence of DNA fragmentation, an index of apoptosis, was not detected in these arrested embryos. A association between time of first cleavage and the rate of early embryonic arrest was observed, with late cleaving embryos more likely to arrest at the 2–4-cell stage than early cleaving embryos. This supports previous observations in bovine (Yadav et al., 1993; Plante et al., 1994; Lonergan et al., 1999, 2000; Ward et al., 2001; Lequarre et al., 2003) and human embryos (Magli et al., 1998, 2001; Fenwick et al., 2002; Racowsky, 2002) that demonstrated increased developmental capability of early cleaving embryos. Furthermore, we hypothesized that these arrested embryos enter a ‘senescence-like state’, which in fetal bovine fibroblasts is associated at the molecular level by the increased abundance of p53 and p66shc (L.A.Favetta et al., unpublished data). Herein, we characterized p53 and p66shc expression levels during different stages of bovine embryo development. Interestingly, p66shc mRNA expression gradually decreases until the blastocyst stage, while the p53 mRNA pattern resembles the common ‘inverted bell-shaped curve’ pattern observed during preimplantation development. Furthermore, 2–4-cell arrested embryos at day 8 post insemination and late cleaving embryos showed an over-expression of p66shc, but not p53. In addition, examination of p53 serine 20 phosphorylation levels, found to be hyper-phosphorylated at the onset of senescence in somatic cells (L.A.Favetta et al., unpublished data), showed no changes among early and late cleaving embryos.
p53 functions as a cell cycle checkpoint regulator that arrests cells entering into the S-phase of the cell cycle. It allows cells to repair the DNA damage or, if incapable of repair, it initiates apoptosis or senescence (Polyak, 1997). The role of p53 during early embryo development is still controversial. Jurisicova et al. (1998) showed an increase in p53 mRNA levels associated with increased apoptosis in mouse preimplantation embryos, but no direct correlations or functional studies have been performed. In undifferentiated mouse embryonic stem cells, p53 nuclear localization is compromised and apoptosis takes place only in a p53-independent manner (Aladjem et al., 1998). By contrast, it is widely accepted that p53 is responsible for apoptosis during mouse postimplantation development (Frenkel et al., 1999). In Xenopus laevis, there is evidence that p53 is necessary for correct embryonic development (Amariglio et al., 1997; Tchang and Mechali, 1999). Our results suggest that p53 has no direct role in early embryonic developmental arrest in cattle. While we found a lack of p53 involvement, it would be interesting to investigate the p53 isoforms, p63 and p73, as possible inducers of early developmental arrest. In fact, both p63–/– and p73–/– knockout mice showed developmental abnormalities, supporting a role for p63 and p73 versus p53 during development (Millis et al., 1999; Yang et al., 1999, 2000). Thus, we speculate that the lack of p53 involvement in embryonic arrest versus a clear role of p53 in senescence in somatic cells may be attributed to the greater embryo-specific relevance of the p53 isoforms, p63 and p73, which might exert p53 functions during early embryonic development.
To strengthen our hypothesis that early developmental arrest is a senescence-like state and that arrested embryos are not undergoing apoptosis, we analysed the morphology of the arrested embryos, looking for signs of apoptosis. In previous studies, Jurisicova et al. (1996) showed that human arrested embryos displayed characteristics of apoptosis at early stages of development, but whether these characteristics are the cause or effect of the embryonic arrest is still unclear (Antczak et al., 1999). In contrast, our study was not able to detect any sign of apoptosis in the bovine 2–4-cell arrested embryos, considering embryos as apoptotic when showing both TUNEL-positive nuclei and morphological signs of apoptosis by PI nuclear staining (Gjørret et al., 2003).
The most novel finding of our study is a possible involvement of p66shc during development and in early developmental arrest. A high expression of p66shc was detected in arrested embryos and in late cleaving embryos, which are more likely to arrest. Low p66shc was detected in the embryos with full developmental capability, at the morula and blastocyst stage. These observations suggest that high p66shc is an index of increased probability of early embryo arrest and poor developmental capability. The precise functions of p66shc are currently being elucidated but it has been shown that p66shc is related to cellular oxidative stress levels and lifespan (Migliaccio et al., 1999) and is downstream to p53 in p53-dependent apoptosis (Trinei et al., 2002). Currently, there are no studies on the role of p66shc in development or evidence of infertility in p66shc–/– mice. Our study is the first to suggest a role for p66shc during early preimplantation development; specifically, high p66shc levels are associated with the induction of embryonic arrest, before the activation of the embryonic genome.
There is increased interest in the literature on gene expression changes associated with early arrest, specifically arrest at maternal–zygote transition, when the activation of the embryonic genome occurs (Schultz, 2002). Before the activation of the embryonic genome, development is regulated by mRNA and proteins synthesized during oocyte growth and maturation (Telford et al., 1990; Brevini-Gandolfi et al., 1999), which is thought to impact embryo development up until the blastocyst stage (Renard et al., 1994). While the majority of the mRNA accumulates during oocyte maturation and is gradually degraded until the activation of embryonic transcription (Paynton and Bachvarova, 1994), p66shc mRNA abundance gradually decreases from the immature oocyte stage to the blastocyst stage, suggesting that the embryos with full developmental capability are those with lower p66shc levels. p66shc mRNA abundance is also higher in the 2–4-cell arrested embryos in comparison to early cleaving 2-cell embryos, collected at 28 hpi. To investigate whether this increase in p66shc was a trigger to the embryo arrest or an effect of the arrest, p66shc mRNA levels were measured in the late cleaving embryos (collected between 29 and 48 hpi), which were more likely to undergo arrest. The finding that these late cleaving embryos not only had high p66shc mRNA, but also protein, suggests that p66shc may be involved in embryonic arrest. Furthermore, high p66shc is also detected in 2–4-cell arrested embryos at day 8 post insemination. Together, these data strengthen the possible role of p66shc as an inducer of early embryo arrest. Further experiments at the oocyte stage would be extremely interesting, as our results suggest that it is probably at the oocyte stage when it is determined which embryos will arrest or fully develop. p66shc might represent a new marker of developmental competence and be used in the future in conjunction with embryo biopsy to screen for embryos that will progress to full development.
Although further studies are necessary to elucidate the functional role of p66shc in early embryonic arrest, our results demonstrate that the developmental potential of IVP embryos is related to the time of first cleavage and that p66shc, but not p53, plays a role in early developmental arrest in in vitro-produced bovine embryos. These results suggest that early embryonic arrest might be a senescence-like state, although with some differences from senescence in somatic cells. While the role of p53 in senescence has been consolidated, p53 is not involved in early embryonic arrest, but there is the common involvement of p66shc. p66shc is a stress-related protein and novel studies empathize the role of oxidative stress in inducing senescence, rather than a critical telomere length. In fact, Stewart et al. (2003) have recently shown that it is the erosion of the telomeric single-strand overhang and the abrogation of its structure, rather than the overall shortening of telomeres, that triggers the senescent state. Furthermore, oxygen free radicals have been shown to be the main cause of single-stranded breaks within telomeric DNA (von Zglinicki et al., 1995; Petersen et al., 1998). Therefore, although further studies are needed to confirm this theory, p66shc might be up-regulated in conditions of oxidative stress, typical of the in vitro culture system, and trigger the senescence-like arrest in early embryos.
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We thank R.Roche and E.Reyes for their technical support and R.Kukreja for the collection of ovaries from the slaughterhouse. Special thanks to James Gilmore and Gabriela Mastromonaco for their critical review of the manuscript. We are grateful to Prof. P.G.Pelicci (Department of Experimental Oncology, European Institute of Oncology, Milan, Italy) for the donation of the p66shc antibody. This research was supported by the Canadian Institutes of Health Research (CIHR), National Science and Engineering Council of Canada (NSERC), the Ontario Graduate Scholarship (OGS) and the Ontario Ministry of Agriculture and Food (OMAF).
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Submitted on January 29, 2004; accepted on March 14, 2004.
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