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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, embryo, ovum
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Apoptosis is a physiological process in which cells die after exposure to normal or pathologic stimuli. Classic biochemical and morphological features of apoptosis are DNA fragmentation, caspase activation, change of mitochondria function, release of cytochrome C, chromatin condensation, cytoplasmic fragmentation, and developmental arrest [19– 23]. Apoptotic cell death occurs in preimplantation mammalian embryos and plays an important role in embryo development. Apoptosis has been observed in bovine embryos after the eight-cell stage [24, 25]. More than 80% of mouse blastocysts had one or more cells undergoing apoptosis [19]. The incidence of cell death in the human blastocyst seems to correlate with cell number and embryo quality [26]. The earliest positive terminal deoxynucleatidyl transferase (TdT)-mediated uridine 5-triphosphate (dUTP) nick-end labeling (TUNEL) signals were detected in porcine embryos derived from nuclear transfer (NT) on Day 5 of culture. The percentage of cells undergoing apoptosis in the NT embryos was higher than that of IVF embryos and increased with time in vitro [27].
To investigate the possibility of the developmental failure in porcine parthenogenetic embryos during in vitro culture, we measured the onset and frequency of apoptosis and morphological changes that conform to the general criteria of apoptotic cell death by TUNEL assay. Apoptosis and the developmental rate were compared between the parthenogenetic embryos (haploid) and in vitro fertilization (IVF) embryos (diploid). The results of these experiments indicated that parthenogenetic embryos undergo a higher degree of apoptotic cell death during culture, relative to IVF.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ovaries were collected from prepubertal gilts at a local abattoir and transported to the laboratory in 0.9% NaCl solution at 30–35°C. Cumulus- oocyte complexes (COCs) were aspirated from antral follicles (3–6 mm in diameter) with an 18-gauge needle fixed to a 10-ml disposable syringe. COCs with uniform cytoplasm and several layers of cumulus cells were selected and rinsed three times in TL-Hepes containing 0.1% (w/v) polyvinyl alcohol (PVA). Approximately 50–70 COCs were transferred into 500 µl maturation medium (TCM 199; 31100035; Gibco, Grand Island, NY). The medium also contained 0.1% PVA (w/v), 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 0.5 µg/ml LH (L-5269; Sigma Chemical Company, St. Louis, MO), 0.5 µg/ml FSH (F-2293; Sigma), 10 ng/ml epidermal growth factor (E-4127; Sigma), 75 µg/ml penicillin G, and 50 µg/ml strepotomycin. The medium had been covered with mineral oil in a four-well Nunclon dish (Nunc, Roskilde, Denmark). The oocytes were matured for 40–44 h at 38.5°C, 5% CO2 in air.
Production of Porcine Preimplantation Embryosfrom Parthenogenetic Activation
After maturation, oocytes were freed from cumulus cells by vigorous vortex for 4 min in TL-Hepes containing 0.1% PVA and 0.1% hyaluronidase. Cumulus-free oocytes with polar body I were exposed to electrical pulses between 0.2-mm diameter platinum electrodes 1 mm apart in fusion/activation medium (0.3 M mannitol, 1.0 mM CaCl2 · H2O, 0.1 mM MgCl2 · 6H2O, and 0.5 mM Hepes). Activation was induced with two DC pulses (1-sec interval) of 1.2 kV/cm for 30 µsec provided by a BTX Electro-Cell Manipulator 200 (BTX, San Diego, CA). The PA embryos were cultured in North Carolina State University-23 (NCSU-23) medium supplemented with 0.4% BSA [28].
Production of Porcine Preimplantation Embryosfrom In Vitro Fertilization
The COCs 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 until sperm were added for insemination. The IVF 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 BSA. For IVF, one 0.1-ml frozen semen pellet was thawed at 39°C in 10 ml DPBS (Gibco) containing 1 mg/ml BSA, 50 IU/ml penicillin K salt, and 50 IU/ml streptomycin sulfate [29]. 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 was added to the fertilization droplets containing the oocytes. At 6 h post insemination, oocytes were washed three times and cultured in 0.5 ml culture medium (NCSU-23 containing 4 mg/ml BSA) in four-well Nunclon dishes (Nunc) at 38.5°C, in 5% CO2 in air.
Apoptosis Assays
The embryos at Days 3, 4, 5, 6, 7, and 8 from PA and IVF were washed three times in PBS/PVP (PBS supplemented with 0.1% polyvinyl- pyrrolidone) and fixed in 4% (v/v) paraformaldehyde solution for 24 h at room temperature. Membranes were permeabilized in 0.1% Triton X-100 in 0.1% citrate solution for 1 h at room temperature.
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. The broken DNA ends of the embryonic cells were labeled with TdT and fluorescein-dUTP. After the reaction stopped, the embryos were washed and transferred into 2 µg/ml DAPI (4', 6-diamidine- 2'-penylindole dihydrochloride; cat. 236 276; Roche) for 30 min at 38.5°C in the dark. The embryos were washed three times and mounted on slides with ProLong antifade kit (cat. P-748, 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 (Promega, 5 µl/50 µl PBS) 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 and DAPI. The numbers of apoptotic nuclei (TUNEL staining) and total numbers of nuclei (DAPI staining) were determined from optical images.
Chromosome Analysis
Chromosome analysis was performed with modification of the procedure described by McCauley et al. [30]. Briefly, on Day 6 of culture, Colcemid solution (0.2 µg/ml; cat.15212-012, Invitrogen Corp., Carlsbad, CA) was added to the embryo culture medium, and the embryos were cultured for 6 h. Then the blastocysts were transferred to Ca2+/Mg2+-free DPBS, and washed two times. The zona pellucida was disrupted by treatment with pronase (5 mg/ml in TCM 199). The embryos were then placed in sodium citrate (0.8% [w/v]) for 3 min, followed by 0.75 M KCl treatment for 2 min. The embryos were transferred onto the slides and a fewer fixing medium (3:1 [v/v] methanol:acetic acid) was dropped onto the slides and the embryos were spread with methanol-acetic acid (1:1[v/v]). The slides were air dried, stained with 2 µg/ml DAPI, and covered by a coverslip and sealed.
Experimental Design and Statistical Analysis
Three experiments were conducted in this study. Each experiment was repeated three or four times. In the first experiment, PA and IVF embryos were evaluated for cleavage on Day 3, and percentage blastocyst on Days 5, 6, 7, and 8. In the second experiment, PA and IVF embryos were collected on Days 3, 4, 5, 6, 7, or 8. The nuclei in these embryos were evaluated for DAPI and TUNEL staining. Cells with only DAPI-stained nuclei (blue color) were considered normal cells, whereas nuclei with TUNEL-stained nuclei (red color) were considered to be DNA-fragmented nuclei. In the final experiment, PA- and IVF-derived blastocysts on Days 5, 6, 7, and 8 were evaluated for nuclear number by DAPI and processed for TUNEL staining. The chromosomes of the blastocysts on Day 6 were analyzed.
Data for 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, percentage of blastocysts, and percentage of apoptotic cells was tested by general linear model procedure (SAS Institute, Cary, NC). Differences among treatment means were determined by using Duncan multiple-range test. All data are expressed as mean ± SEM. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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On Day 3 after activation or IVF, there was a significantly lower cleaved rate for the PA embryos as compared with the IVF embryos (45% vs. 71%, Table 1). The developmental rates to the blastocyst stage were also lower for the PA embryos as compared with the IVF embryos on Days 5, 6, and 7.
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On Day 6 after activation or IVF, development of PA embryos was significantly delayed, compared with the IVF embryo; only 16% of the PA embryos had reached the blastocyst stage, in contrast to 29% of the IVF embryos. Developmental rates of both groups tended to decrease from Day 6 to Day 8 (Table 1).
Onset and Frequency of Apoptosis in Porcine PA and IVF Embryos (Exp. 2)
In this experiment, the fragmentation of DNA was examined in porcine embryos from Day 3 to Day 8 in vitro. The nuclei labeled by TUNEL were counted as single apoptotic nuclei. There was no apoptosis signal (TUNEL) observed on Days 3 or 4. The earliest positive TUNEL signals were detected in the PA embryos on Day 6, 1 day later than IVF embryos (on Day 5, 0.2%; Fig. 1). The percent of apoptosis in the PA embryos tended to increased from Day 7 to Day 8 (14.2% to 29.3%). The percent of apoptosis detected in the PA embryos was higher than IVF embryos except on Day 5 (Table 2).
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Cytoplasmic fragmentation and developmental arrest was examined in embryos as was condensed chromatin in individual nuclei. PA embryos classified as fragmented had irregularly sized blastomeres and fewer nuclei than normal embryos (Fig. 2).
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Apoptosis and Cell Number in Porcine PA and IVF Embryos (Exp. 3)
The mean cell number of IVF blastocysts tended to increase from Day 5 to Day 7. On Day 6, the mean cell number in PA blastocysts (23 ± 1.8) was less than that of IVF blastocysts (29.7 ± 0.8), and the percentage of apoptotic nuclei in PA blastocysts was twice that of the IVF (6% vs. 3%; Table 3, Fig. 3). There was a similar result on Day 7 (7.6% vs. 4.9%), whereas on Day 8, the percentage of apoptotic nuclei in PA blastocysts was six times that of the IVF (18.3% vs. 3.6%). The mean cell numbers in both groups on Day 8 were not significantly different.
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There was no TUNEL staining detected on Day 5. The percent of TUNEL-positive nuclei appeared to remain constant from Day 6 to Day 8 in the IVF group, whereas it appeared to increase from Day 6 to Day 8 in the PA group (Table 3).
Chromosomes of PA Blastocysts
The chromosomes of PA blastocysts were analyzed. Overall, 62.5 % (45 of 72) of the PA blastocysts were haploid, 29.17% (21 of 72) were diploid, and 8.33% (6 of 72) were polyploid (Fig. 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We found that the cytoplasmic fragmented embryos in both IVF and PA groups before Day 6 are not labeled by using TUNEL, but apoptotic staining was detected in most cytoplasmic fragmented embryos after Day 6, and this tended to increase. This suggests that the final death of the cytoplasm fragmented embryos during development is by apoptosis. We also noticed that most of the nuclei that labeled positively with TUNEL appeared in arrested embryos and embryos with condensed nuclei after Day 6. About 75% of embryos fertilized in vitro that had morphological fragmentation also had apoptotic changes [31]. Cytoplasmic fragmentation might represent a regulator or early indicator of apoptosis [27]. Fragments may be equivalent to apoptotic bodies and high levels of fragmentation are associated with reduced blastocyst formation and lower blastocyst cell numbers [31]. When there was extensive fragmentation (>25%), few embryos reached the blastocyst stage, and cell number of the blastocysts was reduced [32]. There are at least four causes involved in apoptosis in PA embryos: activation methods, the lack of a paternal genome, maternally genome and inherited components, and ploidy.
Activation of mammalian oocytes in IVF is normally triggered by sperm penetration. Meiotic metaphase II oocytes can also be activated parthenogenetically by electrical pulses, ethanol, cycloheximide, calcium ionophore, and Sr2+ or combinations of these treatments. Oocyte activation consists of a sequence of morphological and molecular events that include release of Ca2+ and that culminate in the second meiotic division, extrusion of a second polar body, formation of a pronucleus, DNA synthesis, and mitotic cleavage. A double electrical pulse is sometimes used as an activation method; however, double electrical pulses may also have a detrimental effect on embryo development and about 20% of the reconstituted oocytes fragment after parthenogenetic activation [33]. In addition, the degree of apoptosis depends on specific medium and the culture method [24, 34]. So a clear understanding of the pathway of apoptosis in PA embryos may help to improve the suboptimal conditions in the in vitro culture system.
We found that there was a higher percentage haploidy in PA embryos (62.5%), compared with IVF embryos (9.3%). Haploid parthenotes were developmentally retarded (most arrested at the morula stage), and haploid embryos had fewer cells than did diploid embryos [35–38]. Haploidy leads to an increased incidence of apoptosis, and the increased incidence of apoptosis observed in haploid parthenotes is not due to the lack of a paternal genome because diploid parthenotes, without a paternal genome, exhibit a low frequency of apoptosis, comparable with that of IVF embryos [17]. Rather, being haploid must have promoted apoptosis in preimplantation embryos [17]. We found that there was a higher percentage of apoptosis in PA embryos (haploid) after Day 6 in culture, relative to IVF embryos, and there was a trend of an increased incidence of apoptosis during development in vitro. This suggests that apoptosis does not require the paternal genome during preimplantation development, and instead the machinery necessary for apoptosis is inherited from the oocyte. The oocyte contributes not only stored mRNA and proteins for early development but also maternally inherited components, such as mitochondria, for mediating apoptosis.
The onset of apoptosis is related to the activation of the embryonic genome, in which the major events take place at the two-cell stage in mice [39], four-cell stage in humans [31], and 9- to 16-cell stages in cows [25, 40]. However, in NT bovine embryos, DNA fragmentation was first detected at the four-cell stage [25]. Cell death or apoptosis in bovine IVF and NT embryos are dependent on the stage of development [25]. This observation is not consistent with previous studies. We found that the earliest apoptosis in the PA embryos was on Day 6, 1 day later than the IVF embryos, and apoptosis occurred in the fragmented PA embryo arrested at the two-cell stage and IVF embryos arrested at the three-cell stage on Day 6. In porcine NT embryos, the onset of apoptosis was on Day 5, and apoptosis occurred in the embryos arrested at the one-cell stage on Day 5 [27]. We did not find positive TUNEL signals in the porcine IVF, PA, and NT embryos before Day 5. These results indicated that the onset of apoptosis may correlate with the major burst of embryonic genome activation because the activation of the porcine embryonic genome takes place during the four-cell stage.
The number of nuclei and the amount of apoptosis in the embryo are important parameters of embryo development and health [39] because the embryos with a large number of cells are more likely to implant and give rise to live offspring [41]. We observed that the mean cell number in PA blastocysts was less than that of IVF blastocysts from Day 5 to Day 8, but the mean apoptotic cell number in PA blastocysts was higher than that of IVF blastocysts, except on Day 5. These findings suggest that the quality of PA blastocysts was not better than IVF blastocysts from Day 6 to Day 8.
Further research will focus on the transcripts and proteins, which regulate apoptosis, and the maternal mitochondrial function for mediating apoptosis in porcine parthenogenetic embryos.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 573 884 7827; pratherr{at}missouri.edu
Received: 1 December 2003.
First decision: 18 December 2003.
Accepted: 30 January 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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