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Embryo |
Department of Animal Sciences, University of Missouri-Columbia, Columbia, Missouri 65211
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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apoptosis, early development, in vitro fertilization
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The process of apoptotic cell death in preimplantation mammalian embryos has been well described. Apoptosis plays an important role in embryo development [17–19]. Apoptosis occurs during the preimplantation development stage in both in vivo- and in vitro-produced embryos, and it may contribute to embryonic loss. The incidence of apoptosis is higher in bovine blastocysts produced by nuclear transfer (NT) than in embryos produced in vivo [18, 19]. Human preimplantation embryos exhibit high levels of apoptosis and high rates of developmental arrest during the first week in vitro [20]. Thus, apoptosis may contribute to the progressive loss of embryos during the in vitro production procedure.
Research has suggested that a major cause for the level of cell death can be reconciled with the high level of embryo arrest. The developmental competence of embryos likely is already established at the 1-cell stage (zygote). The generation of a healthy zygote is important for understanding the mechanism that causes chromosomal abnormalities during early cleavage stages [21]. Apoptosis has been observed in bovine embryos after the 8-cell stage [22]. More than 80% of in vivo mouse blastocysts on Day 4 or 5 had one or more apoptotic cells [23]. As in the mouse, the incidence of cell death in the human blastocyst seems to correlate with cell number and embryo quality. Blastocysts with fewer cells had a range of TUNEL-positive cells from 0% to 30%, whereas blastocysts with more cells had less than 10% TUNEL-positive cells [17]. However, to our knowledge, porcine embryonic apoptosis and its relation to developmental competence, especially in embryos derived by NT, have not been reported previously.
The objectives of the present study were to investigate the onset and frequency of apoptosis in both NT- and in vitro fertilization (IVF)-derived embryos as well as the morphological changes that conform to the general criteria of apoptotic cell death by TUNEL assay. Morphological changes of apoptosis in relation to preimplantation embryo development and embryo quality were also characterized. Our findings may have important implications for improving porcine preimplantation NT embryo development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All chemicals, unless noted otherwise, were purchased from Sigma Chemical Company (St. Louis, MO). Ovaries were collected from prepubertal gilts at a local abattoir and transported to the laboratory in 0.9% NaCl2 solution at 30–35°C. Cumulus-oocyte complexes (COCs) were aspirated from antral follicles (diameter, 3–6 mm) with an 18-gauge needle fixed to a 10-ml disposable syringe. The COCs with uniform cytoplasm and several layers of cumulus cells were selected and washed three times in Tyrode's lactate (TL)-Hepes containing 0.1% (w/v) polyvinyl alcohol (PVA). Approximately 50–70 COCs were transferred into 500 µl of maturation medium (TCM-199; Gibco, Grand Island, NY) supplemented with 0.1% (w/v) PVA, 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 0.5 µg/ml of luteinizing hormone, 0.5 µg/ml of follicle-stimulating hormone, 10 ng/ml of epidermal growth factor, 75 µg/ml of penicillin G, and 50 µg/ml of streptomycin. The oocytes were matured under mineral oil in a four-well Nunc dish (Nunc, Roskilde, Denmark) for 40–44 h at 38.5°C in 5% CO2 in air.
Preparation of Donor Cells
At 4 days of age, an ear-skin biopsy specimen was obtained from a transgenic pig (402-2) transduced with an enhanced green fluorescent protein (GFP) recombinant retrovirus [24]. The use of these cells for NT was also reported by Park et al. [25]. The tissue was cut into small pieces with fine scissors in PBS containing 0.05% trypsin and 0.02 mM EDTA and was incubated for 30 min at 39°C. The suspension was centrifuged at 300 x g for 10 min, and the cell pellet was resuspended and cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 2 mM L-glutamine, 0.1 mM sodium pyruvate, 75 µg/ml of penicillin G, 50 µg/ml of streptomycin, and 15% (v/v) fetal calf serum (FCS). The cells were passaged three times. Each passage was after approximately a week of culture, and cells were more than 90% confluent at passage. The cells were digested with 0.05% trypsin and 0.02 mM EDTA. The harvested cells were resuspended in 10% dimethyl sulfoxide in culture medium. Then, 50-µl aliquots containing 1500–3000 cells were put into a freezing container (Nalgene, Rochester, NY) and frozen at -80°C overnight. Next, cells were swiftly transferred into liquid nitrogen for long-term storage. Before NT, the donor cells were thawed at 37°C, and 200 µl of FCS were added and cultured for 30 min. Then, 15% FCS was added into 800 µl of DMEM, and the sample was centrifuged at 500 x g for 5 min. Approximately 80% of the cells survived the freeze/thaw process. The supernatant was discarded, and 100 µl of TCM-199 with Hepes were added to resuspend the cells.
Production of Porcine Preimplantation Embryos by NT
After maturation, oocytes were freed from cumulus cells by vortexing for 4 min in TL-Hepes mixed with 0.01% PVA and 0.1% hyaluronidase. Cumulus-free (denuded) oocytes were enucleated by aspirating the first polar body and adjacent cytoplasm in enucleation medium [26]. The medium for micromanipulation consisted of Hepes-buffered TCM-199, 0.3% BSA, and 7.5 µg/ml of cytochalasin B (CB). The medium for injection was the same medium with CB.
A single donor cell was injected into the perivitelline space of enucleated, cumulus-free oocytes to contact the oocyte membrane by using a glass pipette (outer diameter, 30 µm). Injected oocytes were placed between platinum electrodes (diameter, 0.2 mm) that were 1 mm apart in fusion/activation medium, which was made of 0.3 M mannitol, 1.0 mM CaCl2, 0.1 mM MgCl2, and 0.5 mM Hepes. Fusion/activation was induced with 2 DC pulses (interval, 1 sec) of 1.2 kV/cm for 30 µsec with a BTX Electro-Cell Manipulator 200 (BTX, San Diego, CA). The NT embryos were cultured in North Carolina State University-23 (NCSU-23) medium supplemented with 0.4% BSA [27].
Production of Porcine Preimplantation Embryos from IVF
Cumulus-free oocytes were washed three times in IVF medium. Approximately, 35–40 oocytes were transferred into 50-µl droplets of IVF medium covered with mineral oil (Fisher Scientific, Pittsburgh, PA) that had been equilibrated for 40 h at 38.5°C in 5% CO2 in air. The dishes were kept in a CO2 incubator for 20–30 min until sperm were added for insemination. The fertilization medium was a modified Tris-buffered medium consisting of 110 mM NaCl, 0.47 mM KCl, 7.5 mM CaCl2, 0.5 mM sodium pyruvate, 10 mM glucose, 20 mM Tris, 2 mM caffeine, and 2 mg/ml of BSA. For IVF, one 0.1-ml frozen semen pellet was thawed at 39°C in 10 ml DPBS (Gibco) containing 1 mg/ml of BSA, 50 IU/ml of penicillin G, and 50 IU/ml of streptomycin [28].
After washing two times by centrifugation (1900 x g, 4 min), cryopreserved ejaculated spermatozoa were resuspended with fertilization medium to a concentration of 6 x 105 cells/ml. Fifty microliters of the sperm sample were added to the fertilization droplets containing the oocytes. At 6-h postinsemination, oocytes were washed three times and cultured in 0.5 ml of culture medium (NCSU-23 containing 4 mg/ml of BSA) in four-well dishes at 38.5°C in 5% CO2 in air.
Apoptosis Assays
The embryos at Days 3, 4, 5, 6, 7, and 8 from NT and IVF were washed three times with 0.1% polyvinylpyrrolidone in PBS supplemented and fixed in 4% (v/v) paraformaldehyde/PBS solution for 24 h at room temperature. For membrane permeabilization, the embryos were incubated in 0.1% Triton X-100 in 0.1% citrate solution for 1 h.
A TUNEL assay was used to assess the presence of apoptotic cells (in situ Cell Death Detection Kit, TMR red; Roche, Mannheim, Germany). Fixed embryos were incubated in TUNEL reaction medium for 1 h at 38.5°C in the dark and then washed, transferred into 2 µg/ml of DAPI (4',6-diamidine-2'-penylindole dihydrochloride; Roche), and mounted on slides with ProLong Antifade Kit (Molecular Probes, Eugene, OR). The slides were stored at -20°C. As positive controls for TUNEL, fixed embryos were incubated in RQ1 RNase-Free DNase (5 µl/50 µl of PBS; Promega, Madison, WI) for 30 min at 38.5°C in the dark before the TUNEL. Whole-mount embryos were studied with an epifluorescent microscope (Nikon, Tokyo, Japan) by using detection for TUNEL, DAPI, and GFP. The numbers of apoptotic nuclei and total numbers of nuclei were determined.
Experimental Design and Statistical Analysis
Four experiments were conducted in the present study. Each experiment was repeated three or four times. In the first experiment, NT and IVF embryos were evaluated for cleavage on Day 3 and for the percentage blastocyst and nuclear number on Day 6 after IVF. In the second experiment, IVF and NT embryos were evaluated on Days 3, 4, 5, 6, 7, or 8 for cytoplasmic fragmentation and developmental arrest. The nuclei in these embryos were evaluated for condensed chromatin and apoptosis. In the third experiment, cytoplasmic fragmented embryos, arrested embryos, and embryos with condensed chromatin were evaluated in embryos derived by IVF or by NT. In contrast to the second experiment, here the data were collected on a per-embryo basis. In the fourth experiment, morphologically normal blastocysts were collected on Day 5, 6, 7, or 8 and then processed for TUNEL staining.
Embryos were classified as follows: 1) cytoplasmic fragmented embryos, embryos with fewer nuclei than cytoplasts and with irregularly size blastomeres on Day 3, 4, 5, 6, 7, or 8 of culture; 2) arrested embryos, embryos that were still at the 1-cell stage on Day 3, 4, 5, 6, 7, or 8 of culture; 3) condensed embryos, embryos in which the embryonic chromatin condensed into a single "speck" on Days 3, 4, 5, 6, 7, or 8 of culture; 4) DNA fragmented cells, cells with TUNEL-stained nuclei (red color) in addition to the DAPI staining; and 5) normal cells, cells with only DAPI-stained nuclei (blue color). Unfertilized oocytes in the IVF group that were at the 1-cell stage were differentiated from arrested fertilized zygotes after staining with DAPI and confirmation of fertilization. Polyspermic embryos could not be differentiated from monospermic embryos after the 2-cell stage, so they could not be eliminated from the data set.
Data regarding the percentage of apoptotic blastomeres were transformed by ArcSin to overcome heterogeneity of variance. The statistical significance between days of embryo development and treatment effects of cleavage rate, total cell number, and percentage of blastocysts as well as fragmented, arrested, condensed embryos was tested by SAS GLM procedure [29]. Differences among treatment means were determined by using the Duncan multiple-range test. All data are expressed as least-square mean ± SEM. Differences were considered to be significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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On Day 6, almost twice as many of the IVF embryos cleaved as compared to the NT embryos (71% vs. 42%). The developmental rates to the blastocyst stage were also higher for the IVF embryos as compared to the NT embryos (28% vs. 11%; data not shown). However, the number of nuclei in each blastocyst was not different between the groups. Thus, whereas the NT embryos developed to the blastocyst stage at a lower rate, these blastocysts had the same number of nuclei as the IVF embryos and appeared to be morphologically normal.
Cytoplasmic Fragmentation and Developmental Arrest of NT and IVF Embryos (Experiment 2)
In this experiment, IVF and NT embryos were evaluated on Day 3, 4, 5, 6, 7, or 8 for abnormal development. Cytoplasmic fragmentation and developmental arrest were examined in embryos, as was condensed chromatin in individual nuclei (Table 1). Embryos classified as fragmented had irregularly sized blastomeres and fewer nuclei than normal embryos (Fig. 1). Embryos arrested at the 1-cell stage were also noted (Fig. 2). The nuclei with condensed chromatin had a nuclear envelope in a ring or in a single "speck," and the chromatin was dispersed (Fig. 3).
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On Day 3, 110 NT embryos were evaluated (Table 1). Of these, 37.7% had cytoplasmic fragmentation, 32.6% were arrested at the 1-cell stage, and 29.7% appeared to be normal. On Day 6, out of 143 NT embryos evaluated, 44.3% had cytoplasmic fragmentation, 15.6% were arrested at the 1-cell stage, and 40.1% appeared to be normal. On Day 8, out of 114 NT embryos evaluated, 47.2% had cytoplasmic fragmentation, 17% were arrested at the 1-cell stage, and 35.8% appeared to be normal. The percentage of cytoplasmic fragmented embryos numerically increased with time in culture; however, it should be noted that the experiment was designed to evaluate NT versus IVF embryos. The level of cytoplasmic fragmentation of NT embryos was higher than that of IVF embryos on Days 4, 5, and 6.
Chromatin condensation was observed in most of the cytoplasmic fragmented and arrested embryos. On Day 3, only 1.5% of total nuclei of the NT embryos were condensed, whereas no condensed chromatin was observed in the IVF embryos. The percentage of nuclei that had condensed chromatin tended to increase with time, except for Day 5. The percentage of the NT-derived nuclei that had condensed chromatin was higher than that of IVF-embryos on Days 5, 7, and 8.
No TUNEL-positive nuclei were observed before Day 5. On Day 5, TUNEL-positive nuclei could be detected, and the percentage tended to increase with time and was higher for the NT nuclei as compared to the IVF nuclei (Table 1).
TUNEL on a Per-Embryo Basis in IVF- and NT-Derived Embryos (Experiment 3)
In this experiment, TUNEL staining was performed on morphologically abnormal embryos on Days 3, 4, 5, 6, 7, and 8 (Table 2 and Figs. 1– 3). No apoptotic cells were detected in cytoplasmic fragmented embryos before Day 5. On Day 5, the percentage of apoptotic nuclei was 12% in the fragmented NT embryos and zero in the fragmented IVF embryos. This percentage was higher on Days 6, 7, and 8, and no difference was observed between the two groups. On Day 5, 1.2% of nuclei were apoptotic in arrested NT embryos. No apoptotic nuclei were found in arrested IVF embryos. This number tended to increase with time in both groups and was higher in NT than in IVF embryos on Day 8. The percentage of TUNEL-positive cells in embryos containing condensed chromatin was zero before Day 5. On Days 5 and 6, the percentage of positive TUNEL staining was higher in the NT embryos as compared to the IVF embryos.
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Apoptosis in Morphologically Normal Blastocysts (Experiment 4)
Embryos were morphologically evaluated on Days 5, 6, 7, and 8. The percentage of normal blastocysts was higher in IVF blastocysts than in NT blastocysts for each day (Table 3). Between the different days, it appears that the percentage of blastocysts in the IVF embryos increased from Day 5 to Day 6 and reached a plateau on Days 7 and 8. In contrast, the percentage of blastocysts in NT embryos reached a peak on Day 6. Both total cell number and percentage of apoptotic nuclei tended to increase from Day 5 to Day 7. Whereas the number of nuclei in each treatment group was similar, except for Day 7, the percentage of TUNEL-positive nuclei was higher for the NT blastocysts on Days 6 through 8. No TUNEL staining was detected before Day 5. The percentage of TUNEL-positive nuclei appeared to remain constant from Day 6 to Day 8 in the IVF group, but it appeared to increase from Day 6 to Day 8 in the NT group (Table 3 and Fig. 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Morphological Evidence of Apoptosis in Porcine Preimplantation NT Embryos
We observed cytoplasmic fragmentation in IVF embryos. We also showed that cytoplasmic fragmentation and developmental arrest were the main morphological characteristics of embryos undergoing apoptosis. Many porcine NT embryos were unable to proceed through preimplantation development. Cytoplasmic fragmentation was detected in 37.7% of the embryos on Day 3 of in vitro culture.
The relationship between cytoplasmic fragmentation and DNA fragmentation was further investigated. It is interesting that, although fragmented embryos were found among two experimental groups before Day 5 of in vitro culture, no apoptotic signal was observed. We found that the cytoplasmic fragmentation occurred first, followed by the DNA fragmentation. Cytoplasmic fragmentation might represent a regulator or early effector phase of apoptosis. We also investigated the relationship between developmental arrest and apoptosis. The results indicate that embryo developmental arrest is associated with subsequent apoptosis, although cytoplasmic fragmentation does not occur. We further observed condensation of chromatin in most of the fragmented and arrested embryos. These results suggest that DNA fragmentation occurred after the condensation of chromatin.
A relationship is possible between embryonic morphology and developmental competence. We suggest that morphological abnormalities, including cytoplasmic fragmentation, arrest, and nuclear condensation, are typical morphological changes of embryos undergoing apoptotic cell death. As a result, selecting different grades of embryos is essential to improve embryo production using in vitro culture.
Evidence of Apoptosis in Blastocysts
The percentage of NT embryos that form a blastocoele cavity was assumed to increase from Day 5 to Day 6 and then reach a plateau for another 2 days. Interestingly, the percentage of blastocysts in the NT group peaked on Day 6 and then decreased. This decrease may indicate that these embryos were very fragile and might have been dying. In fact, the number of apoptotic nuclei increased over time in the NT group, whereas it remained unchanged in the IVF group. Again, this suggests that the NT embryos were undergoing cell death, resulting in a decrease in the percentage of blastocysts.
These results imply that the quality of the NT blastocysts is not better than that of the IVF blastocyst and that the quality of late blastocysts is not better than that of early blastocysts. Our results demonstrate the usefulness of the TUNEL assay for evaluating the quality of blastocysts. We suggest that in vitro-produced porcine blastocysts on Day 6 or normal embryos before Day 6 are optimal for embryo transfer, even though apoptotic nuclei are found in blastocysts at this time. In addition, a longer period of culture in vitro may increase the percentage of apoptotic nuclei in blastocysts.
Interestingly, a mosaic expression of the enhanced GFP has been shown in the NT embryos [25]. Similarly, in the cells from the animals cloned by Park et al. [25], the mosaic expression observed was the same as in the donor cells. In that paper, it was suggested that the mosaic expression might be linked to cell death. However, in the present study, we show no correlation with enhanced GFP expression and TUNEL staining. Thus, the mosaic expression observed by Park et al. likely is a result of normal differential expression of genes within a single embryo at a single stage.
Possible Causes of Apoptosis in Preimplantation NT Embryos
Every cell has an apoptotic pathway. The propensity to apoptosis is continuously counterbalanced in the cell by genes stimulating cell survival and proliferation. On induction by an appropriate trigger, the cell activates or stops the repression of gene products responsible for control of the suicidal mechanism [30]. Genes are involved in cell death (e.g., Bad and Bcl-xS) and in cell survival (Bcl-2) [31].
In the present study, the earliest positive TUNEL signals were detected in the morphologically abnormal NT embryos on Day 5 of in vitro culture, and no apoptosis was observed on Days 3 and 4. It is not clear why no apoptosis was observed earlier. It is known that chromatin structure changes during early development [32]. The 4-cell stage is when the major onset of gene expression begins. Also, it is not clear if the nuclei in the NT embryos were fully reprogrammed and synchronous with the cytoplasm of the activated oocyte. Furthermore, there appears to be an interaction between DNA methylation and histone acetylation that could impact imprinted genes.
Some changes occur in the expression of parentally imprinted genes after NT [33, 34]; that is, transcription was silenced after NT and resumed at the late 1-cell stage. Tri-butyl-phosphate was displaced and subsequently accumulated at the early and the late 1-cell stage [35]. The DNase I sensitivity was increased and then decreased at the early and late 1-cell stage [35]. So, although these changes proceed gradually, some change could be an appropriate trigger to induce the expression of genes involved in cell death so that apoptosis occurs.
In the present study, morphologically abnormal NT embryos exhibited a very high degree of apoptosis as compared to IVF embyros. The bovine IVF embryos did not express the Bcl-xS transcript, but some fragmented embryos did [31]. A high expression of BCL-2 is found at the cleavage and blastocyst stages. However, low expression of BAX is found at cleavage stages and in blastocysts of good morphology, and BAX expression is increased in blastocysts with extensive fragmentation [31]. These observations confirm that embryos undergoing apoptosis express several apoptosis-related genes during preimplantation embryo development, with severe changes when apoptosis is activated.
On the other hand, DNA damage could be mediated by the mitochondrial pathway in the cytoplasm. The members of the activated caspase family of proteases can induce this pathway. Damage to mitochondria by different stimuli can result in the release of cytochrome c, which together with Apaf-1 and dATP lead to the recruitment and activation of pro-caspase. The pro-caspase is present in several intracellular compartments, including the mitochondrion, Golgi, cytosol, and nucleus [36]. What can initiate the mitochondrial pathway in the reconstructed NT embryo? What role do the mitochondria have in both the donor cell and oocytes in apoptosis of the reconstructed NT embryo? These questions remain unsolved.
In summary, the porcine NT embryos had a lower development rate and a lower cleavage rate during in vitro development as compared to IVF embryos. Apoptotic cell death was a main cause. The earliest positive TUNEL signals were detected in the NT embryos on Day 5 of in vitro culture. The percentage of apoptosis in the NT embryos increased as the culture time increased. Morphologically abnormal changes of the embryo during early development were related to apoptosis. The cytoplasmic fragmentation, developmental arrest, and nuclear condensation were typical characteristics of embryos undergoing apoptosis. The TUNEL assay can be used as a way of selecting different grades of embryos and evaluating the quality of blastocysts. Some special changes in the nucleus and cytoplasm in NT embryos during preimplantation development could trigger the apoptotic pathway. It may be possible to improve the developmental potential of NT embryos by preventing activation of the apoptotic pathway, especially during stages of preimplantation embryo development.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Randall S. Prather, 920 East Campus Drive, E125D Animal Science Research Center, University of Missouri-Columbia, Columbia, MO 65311-5300. FAX: 573 884 7827; pratherr{at}missouri.edu
3 Visiting Scientist from Life Science and Biotechnology Research Center, Northeastern Agricultural University, Harbin, P.R. China
Received: 6 February 2003.
First decision: 5 March 2003.
Accepted: 28 March 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 2002 295:1089-1092This article has been cited by other articles:
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  P. M. Misica-Turner, F. C. Oback, M. Eichenlaub, D. N. Wells, and B. Oback Aggregating Embryonic but Not Somatic Nuclear Transfer Embryos Increases Cloning Efficiency in Cattle Biol Reprod, February 1, 2007; 76(2): 268 - 278. [Abstract] [Full Text] [PDF]
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 B. Mateusen, A. Van Soom, D. G D Maes, I. Donnay, L. Duchateau, and A.-S. Lequarre Porcine embryo development and fragmentation and their relation to apoptotic markers: a cinematographic and confocal laser scanning microscopic study Reproduction, April 1, 2005; 129(4): 443 - 452. [Abstract] [Full Text] [PDF]
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 R. S. Prather, P. Sutovsky, and J. A. Green Nuclear Remodeling and Reprogramming in Transgenic Pig Production Experimental Biology and Medicine, December 1, 2004; 229(11): 1120 - 1126. [Abstract] [Full Text] [PDF]
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W. E. Thompson, E. Asselin, A. Branch, J. K. Stiles, P. Sutovsky, L. Lai, G.-S. Im, R. S. Prather, S. C. Isom, E. Rucker III, et al. Regulation of Prohibitin Expression During Follicular Development and Atresia in the Mammalian Ovary Biol Reprod, July 1, 2004; 71(1): 282 - 290. [Abstract] [Full Text] [PDF]
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Y. Hao, L. Lai, J. Mao, G.-S. Im, A. Bonk, and R. S. Prather Apoptosis in Parthenogenetic Preimplantation Porcine Embryos Biol Reprod, June 1, 2004; 70(6): 1644 - 1649. [Abstract] [Full Text] [PDF]
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