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Embryo |
Department of Anatomy and Physiology,2 Royal Veterinary and Agricultural University, 1870 Frederiksberg C, Denmark
Department of Farm Animal Health,3 Faculty of Veterinary Medicine, Utrecht University, 3584 CL Utrecht, The Netherlands
Department of Clinical Studies, Reproduction,4 Royal Veterinary and Agricultural University, 1870 Frederiksberg C, Denmark
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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apoptosis, developmental biology, early development, embryo, in vitro fertilization
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Morphological characteristics of apoptotic cell death, such as chromatin condensation and marginalization and nuclear fragmentation by karyorhexis [4], are visible in unarrested morula and blastocyst stage embryos produced both in vivo and in vitro. Such nuclear changes are observed in 70%–80% of all in vitro-produced blastocysts from mice [12] and humans [13] and in practically all blastocysts from cattle [14], and the presence of these changes has been taken as evidence of apoptotic activity. Ultrastructural studies of blastocysts have revealed extensive chromatin and cytoplasmic condensation, nuclear and cell fragmentation with intact organelles, and phagocytosis [15, 16].
The TUNEL reaction [17] enables in situ detection of apoptotic cells by labeling of extensive oligonucleosomal DNA fragmentation generated by endogenous DNase activity during the apoptotic process. Initial application of this reaction assay to preimplantation embryos [18] has opened a new line of research, and it has been used for observation of apoptotic cell death in cleavage, morula, and blastocyst stages from many species [6, 8–10, 14, 19–27]. Unfortunately, the TUNEL reaction fails to determine how DNA degradation is generated, because nuclei of cells undergoing necrosis are also labeled [28] and inadequate tissue handling may induce sufficient DNA damage to generate labeling of apparently normal nuclei [29]. Apoptosis may not always be associated with extensive DNA degradation [30, 31], and when it occurs, DNA degradation appears to be a relatively late event in the apoptotic process [32]. The morphological appearance of the TUNEL reaction in necrotic cells is, however, somewhat different because the mode of nuclear disintegration is by karyolysis rather than by karyorhexis, as seen in apoptosis [4, 33]. Therefore, morphological evaluation must be performed when the apoptotic mode of cell death is to be quantified, but more than one feature of apoptosis must be observed for correct identification of the process [33]. Variation in the assessment of apoptotic incidence in preimplantation embryos may arise because of discrepancies in definitions. Some researchers regard TUNEL-positive nuclei as apoptotic without implying morphological evaluation, others regard both TUNEL-positive nuclei and nuclei with apoptotic morphology but without TUNEL reaction as apoptotic, and others rely only on morphological changes for quantification of apoptosis. Therefore, results are not always directly comparable among studies.
The chronology of onset of apoptotic cell death in preimplantation embryos has been debated because various characteristics of this complex process, such as cell and nuclear fragmentation, DNA degradation, and phagocytosis, emerge at different developmental stages. There is little evidence of apoptotic cell death with detectable DNA degradation prior to compaction in unarrested human and mouse embryos with normal morphological appearance that have been produced either in vivo or in vitro [12, 24, 26], but signs of apoptosis have been observed at the eight-cell stage in similar in vitro-produced bovine embryos [6, 14]. However, in embryos arrested in development, displaying no mitotic activity within 24 h, characteristics of apoptosis can be observed at earlier stages [18]; whether these characterstics are a cause or a result of embryonic arrest is still unclear [34]. Because different markers of apoptotic cell death are needed concurrently for exact identification [33], a thorough knowledge of their occurrence and regulation is crucial for adequate evaluation of apoptosis in preimplantation embryos. To our knowledge, this is the first report of the independent chronological appearance of both apoptotic nuclear morphology and DNA degradation during bovine preimplantation development in vivo and of direct comparisons to similarly staged embryos developed in vitro.
In this study, we investigated the chronological occurrence of two key apoptotic markers: 1) changes in nuclear morphology, such as nuclear and chromatin condensation and nuclear fragmentation, typical for apoptosis, and 2) DNA degradation detectable by the TUNEL reaction. We assessed these markers at various preimplantation stages in bovine embryos with expected developmental kinetics that were produced either in vivo or in vitro. We wanted to determine 1) whether the markers could be found at different developmental stages and whether in vitro production (IVP) alters the chronological appearance and ratios of embryos displaying these markers, thereby providing information about the underlying cell death machinery, and 2) whether any differences in the incidence and distribution of apoptotic cell death in bovine morulae and blastocysts produced either in vivo or in vitro could be observed using a scoring system encompassing both markers simultaneously. These results can be used a reference for future mechanistic studies and should stimulate the use of similar combinations of markers in other experiments.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In vivo-produced embryos were collected from normal cyclic Holstein-Friesian heifers (n = 24), which were synchronized and superovulated with postponement and monitoring of the LH surge as described previously [35] with some modification. Dominant follicle ablation was performed on Day 8 (synchronized estrus = Day 0), and subsequent superovulation (Day 10) was instigated with sheep FSH (Ovagen; ICP, Auckland, New Zealand), which was given i.m. twice daily for 4 days in a decreasing regimen (total dose of 299 IU NIH-FSH-S1). A 3-mg progesterone ear implant (Crestar; Intervet International BV, Boxmeer, The Netherlands) inserted concurrently with the first administration of FSH and was removed 96 h after insertion, and 21 mg of a GnRH analogue (buserelin acetate, Receptal; Intervet) was given i.m. 12 h after the last FSH injection. All animals were inseminated 12–14 h after GnRH administration with frozen-thawed semen from a known fertile bull according to standard procedures. The LH surge was monitored as previously described [36] using a validated RIA method [37]. The LH surge was observed 
2 h after GnRH administration, and ovulation was expected 24 h after the surge.
All animals were killed, and embryos were collected as previously described [36]. Embryonic developmental stage and general morphologic appearance were assessed by stereo microscopy. Embryos at different developmental stages were collected at the following time points postovulation (p.o.): 2-cell stage at 38 h p.o., 3- to 8-cell stage at 48 h p.o., 9- to 16-cell stage at 81 h p.o., morula stage at 131 h p.o., and blastocyst stage at 168 h p.o. Collected embryos were washed briefly in PBS with BSA, fixed for 1 h at room temperature in 4% paraformaldehyde (PFA; Merck, Darmstadt, Germany) in PBS, transferred to 1% PFA, and stored at 4°C until further analysis.
In Vitro Embryo Production
In vitro-produced embryos were harvested from four experimental replicates following standard procedures as previously described [38, 39], with all incubations performed in a Heraeus incubator at 38.8°C in humidified air with 5% CO2. Oocytes were derived from cattle abattoir ovaries, and in vitro-matured oocytes were used for in vitro fertilization (IVF) according to standard procedures using frozen-thawed semen from a known fertile bull. Quality of semen used for embryo production both in vivo and in vitro was regarded as equal with respect to support of subsequent embryo development. Following IVF, 20–25 inseminated oocytes were added to culture drops and cocultured with bovine oviduct epithelial cells until harvesting.
Embryonic developmental stage and general morphologic appearance were assessed by stereo microscopy, and embryos were collected at the following time points postinsemination (p.i.): 2-cell stage at 32 h p.i., 3- to 8-cell stage at 40 h p.i., 9- to 16-cell stage at 100 h p.i., morula stage at 117 h p.i., and blastocyst stage at 160 h p.i. Collected embryos were washed and fixed as above, and specimens were stored for no longer than 2 wk before further processing.
TUNEL and Confocal Microscopy
Nuclei with DNA degradation were detected using a cell death detection technique based on the TUNEL principle [17] with fluorescein-conjugated dUTP as described previously [14, 26] with minor modifications. Fixed embryos were subjected to TUNEL reaction (In Situ Cell Death Detection Kit; Roche, Hvidovre, Denmark). Extensive DNA fragmentation was induced in positive controls by incubation in 50 U/ml DNase (RQ1; Promega; Bie & Berntsen, Rødovre, Denmark) prior to the TUNEL reaction, and negative controls were generated by omitting terminal transferase from the reaction. Labeled embryos were all incubated in 0.1 mg/ml of RNase A (Sigma, St. Louis, MO), and DNA was counterstained with 10 µg/ml propidium iodide (PI; Sigma). Embryos at the two-cell to morula stages were mounted on glass slides in 10–15 µl Flouroguard anti-fade (BioRad, Hercules, CA) under coverslip compression, but to conserve spherical morphology blastocysts were taken through an increasing gradient of Vecta-Shield anti-fade (Vector Laboratories, Burlingame, CA) and mounted in pure Vecta-Shield with 0.05 µg/ml PI within a plastic ring, placed between the glass slide and coverslip, to prevent blastocyst compression. Slides were stored at 4°C for up to 7 days before fluorescence microscopic evaluation.
All specimens were examined using a DM-RB fluorescence microscope (Leica Microsystems AG, Wetzlar, Germany) with 16/40x PL Fluotar/0.75 oil objectives and appropriate filters for red (PI) and green (fluorescein) fluorescence detection. Scoring of nuclei in two-cell to morula stage embryos was performed according to the criteria described below. Selected two-cell to morula stage embryos and all blastocysts were subsequently subjected to confocal laser-scanning microscopy on a Leica TCS4D microscope (Leica Laser Technik, GmbH, Heidelberg, Germany) using an argon/krypton laser at 488 and 568 nm and two-channel scanning for detection of fluorescein isothiocyanate and PI, respectively. Complete Z series of 20–25 optical sections at 3- to 4-µm intervals were acquired from each embryo using Leica Scanware software. With this sectioning interval, all nuclei appeared on at least two consecutive images, thereby assuring that all nuclei of an embryo were registered. Image stacks were reconstructed with a Silicon Graphics octane computer (SGI, Mountain View, CA) equipped with an Imaris image analysis software package (Bitplan AG, Zurich, Switzerland), and reconstructed confocal images were used for scoring of nuclei in the trophoblast (Tb) and inner cell mass (ICM) separately in each blastocyst. Allocation of nuclei to each embryonic compartment was based on position in the reconstructed images, and nuclei of Tb cells covering the ICM, i.e., the polar Tb, were for practical reasons included in the ICM, and the remaining mural trophoblast constituted the Tb compartment.
Scoring of Nuclei
The total number of nuclei was counted during scoring, and nuclear morphology was assessed on the basis of PI staining and was scored as being normal, condensed (i.e., pyknotic), or fragmented. Normal nuclei displayed loose reticulated chromatin content and sharp delineations, whereas condensed nuclei exhibited stronger PI staining of compacted chromatin in a decreased volume when compared with normal nuclei within the same embryo (Fig. 1A). Condensed nuclei displayed sharp delineations, were often spherical in shape, and contained either a homogenous chromatin content or chromatin aggregated in marginalized clumps along the nuclear envelope, which sometimes gave the nucleus a lobulated appearance. Fragmented nuclei had two or more condensed chromatin fragments, also with sharp delineations (Fig. 1B). A cluster of nuclear fragments confined in an area comparable to or smaller than the volume of a normal nucleus was regarded as originating from a single nucleus. Conversely, when two fragments were separated by a distance of at least the diameter of an average nucleus, they were regarded as originating from different nuclei. Some nuclei displayed a different mode of disintegration; they had increased PI staining intensity but lacked a reduction in volume and had an unclear or fluffy delineation (and were often TUNEL positive; Fig. 1C), and they were sometimes fragmenting into numerous minute elements in an expanded volume. These nuclei were not classified as apoptotic because they could represent necrotic or other types of cell death, but they were included for calculation of the total number of nuclei.
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DNA degradation was assessed by observation of a distinct TUNEL reaction of chromatin, and the nuclear origin of labeled material was verified by colocalization with PI staining. According to the criteria described above, nuclei were classified as 1) normal, 2) displaying morphological characteristics of apoptosis (nuclear condensation with or without fragmentation; +M), 3) displaying the biochemical characteristics of apoptosis (TUNEL positive; +T), 4) displaying both morphological and biochemical characteristics of apoptosis (M&T), or 5) displaying one or both of the characteristics of apoptosis (M+T) (Fig. 1D). In this study, nuclei were only regarded as apoptotic if in addition to being TUNEL positive they also displayed apoptotic morphology, i.e., they were allocated to the M&T subset of nuclei. Indices (percentages) based on total number of nuclei were calculated for each subset respectively in morulae and separately for the ICM and Tb compartment and in total in blastocysts. To validate the scoring procedure, nuclei of 10 randomly selected blastocysts were reassessed three times and variation between repeated scores was <8% for all parameters.
Statistical Analysis
Ratios of in vivo-produced and in vitro-produced embryos at different stages displaying at least one nuclus with apoptotic morphology and TUNEL reaction were compared by chi-square tests, whereas indices of +M, +T, M&T, and M+T were compared using either unpaired Student t-tests with a Welch correction after the Kolmogoro-Smirnov test for normality or using Mann-Whitney U-tests when the data did not follow a normal (Gaussian) distribution. Correlation analysis between cell numbers and incidence of apoptotic nuclei in blastocysts was performed by a Pearson test assuming normal distribution of data. All tests were performed with a GraphPad InStat 3.05 statistical software package (Graph Pad Software Inc., San Diego, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A total of 213 preimplantation in vivo embryos and 201 preimplantation in vitro embryos were analyzed; the distribution of embryos at various developmental stages is presented in Table 1. Embryos subjected to preincubation in DNase (positive controls) displayed the TUNEL reaction in all nuclei, whereas when terminal transferase was omitted (negative controls) no labeling of any nuclei was observed (data not shown). Apoptotic morphology was not observed in any two-cell embryos (Table 1), but it was first observed as nuclear condensation in a six-cell in vitro embryo and an eight-cell in vivo embryo. Thus, apoptotic morphology was virtually not observable prior to the fourth cell cycle. A condensed nucleus of an 8-cell in vitro embryo displayed some degree of nuclear lobulation, but a classic apoptotic pattern of nuclear fragmentation was not observed until the 9-cell stage in in vitro embryos and the 21-cell stage in in vivo embryos. Thus, nuclear condensation could be observed at earlier developmental stages than nuclear fragmentation and appeared almost simultaneously in vitro and in vivo, whereas nuclear fragmentation occurred earlier in vitro than in vivo.
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The earliest observation of the TUNEL reaction was in a condensed but not fragmented nucleus of a six-cell in vitro embryo, which represented the earliest observation of apoptosis in the present study. The first observation of the TUNEL reaction in vivo was made in a condensed but not fragmented nucleus of a 21-cell embryo, whereas the first observations of nuclei displaying both nuclear condensation and fragmentation in combination with the TUNEL reaction were made in a 19-cell in vitro embryo and a 28-cell in vivo embryo. TUNEL reactions in nuclei without apoptotic morphology were first observed in an 18-cell in vitro embryo and a 60-cell in vivo embryo. However, apoptotic morphology and the TUNEL reaction were observed in only a few embryos prior to the 16-cell stage, and when comparing the ratios of in vivo and in vitro embryos displaying at least one nucleus with either apoptotic morphology or apoptotic morphology and the TUNEL reaction (Table 1), no significant differences were observed at any stage (P > 0.1).
When nuclei with apoptotic morphology (with or without the TUNEL reaction) were observed in precompaction embryos, it predominantly occurred in marginalized blastomeres (Fig. 1A). When such marginalized blastomeres were observed in the perivitteline space of postcompaction embryos, they were considered evidence of cell extrusion. These blastomeres were relatively large and bulky and often contained highly condensed nuclei, with or without fragmentation and the TUNEL reaction. However, some of these extruded blastomeres contained nuclei displaying a morphology that was not classified as apoptosis. Marginalization of blastomeres was observed from the 9- to the 16-cell stage in both in vitro embryos and in vivo embryos. At all embryonic stages examined, cytoplasmic fragments of various sizes with diffuse PI staining but no observable chromatin content were seen in both in vivo and in vitro embryos. Various cell changes indicating apoptosis are presented in Figure 1G.
Incidences of Apoptotic Changes in Morulae and Blastocysts
In morulae and blastocysts, 84.1% and 87.7% of TUNEL-positive nuclei (+T) observed in vivo and in vitro, respectively, also displayed apoptotic morphology (M&T). Thus, according to our criteria 15.9% and 12.3% of the TUNEL-positive nuclei were not regarded as apoptotic. At these stages, only 66.8% and 57.1% of nuclei with apoptotic morphology (+M) also displayed the TUNEL reaction (M&T) in vivo and in vitro, respectively. Thus, 33.2% and 42.9% of the nuclei with apoptotic morphology in vivo and in vitro, respectively, were not regarded as apoptotic.
In vivo morulae contained more nuclei (77.3 ± 6.6) than their in vitro counterparts (43.3 ± 3.4; P < 0.0001), but when comparing percentages of nuclei displaying apoptotic morphology (+M; 3.4% ± 0.7% in vivo vs. 6.1% ± 1.4% in vitro), TUNEL reaction (+T; 2.4% ± 0.6% in vivo vs. 3.1% ± 0.1% in vitro), apoptosis (M&T; 2.0% ± 0.5% vs. 2.9% ± 0.9% in vitro), and one or both of the features (M+T; 3.9% ± 0.7% in vivo vs. 6.5% ± 1.5% in vitro), no difference were observed (P > 0.05 for all parameters). At the blastocyst stage, no differences in numbers of cells in the ICM, Tb, or the total blastocyst were observed between in vivo and in vitro embryos, but the percentages of nuclei scored as +M, +T, M&T, and M+T were all higher in the ICM than in the Tb regardless of production system (P < 0.01; Fig. 1, E and F). However, several differences were observed between in vivo and in vitro blastocysts (Table 2). The percentages of +M, +T, M&T, and M+T nuclei were all significantly lower for in vivo total blastocysts than for in vitro total blastocysts (+M, P < 0.0005; +T, P < 0.05; M&T, P < 0.05; M+T, P < 0.001), and these differences were the result of significant differences in percentages for the ICM (+M, P < 0.001; +T, P < 0.05; M&T, P < 0.05; M+T, P < 0.001), whereas no significant differences were observed for the Tb (P > 0.05 for all parameters).
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No significant correlation between total numbers of nuclei and the percentages of apoptotic nuclei were observed in blastocysts produced either in vivo (R2 = 0.0186, P = 0.4342) or in vitro (R2 = 0.1109, P = 0.0671) (data not shown). However, when comparing data for the ICM and Tb separately, a significant correlation between cell number and apoptotic incidence was observed in the ICM of in vitro embryos (R2 = 0.1723, P = 0.0202) but not in vivo embryos (R2 = 0.0077, P = 0.6170) (Fig. 2). No such correlation was evident in the Tb of in vivo embryos (R2 = 0.003239, P = 0.7454) or in vitro embryos (R2 = 0.01370, P = 0.5307) (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Expression of different morphological and molecular elements of the apoptotic program may be developmentally regulated in mammalian embryos. Cellular fragmentation has been reported in the earliest stages of development both here and in other studies [2, 24], but it is controversial whether this feature as such represents true apoptotic activity [34]. However, the fragmentation of cells into membrane-bound elements is a key feature of the apoptotic process [3], and its occurrence in cleavage stages, without other signs of apoptosis, may represent a specific activation of a subsection of the apoptotic machinery responsible for this characteristic. In this study, extensive nuclear condensation became apparent at the six-cell stage concurrently with the first observation of DNA degradation in in vitro embryos. This observation is consistent with those of previous studies of bovine in vitro embryos, where apoptosis was first observed at the eight-cell stage as indicated by either apoptotic morphology and the TUNEL reaction [14] or the TUNEL reaction alone [6, 8]. This is the first study to investigate the comparable chronological appearance of apoptotic markers in both in vivo-produced and in vitro-produced bovine embryos. IVP in this species seems to affect the developmental regulation of DNA degradation, because this apoptotic element was not detectable before the 21-cell stage in vivo. The occurrence of DNA degradation in vivo is more consistent with observations in other species, where apoptotic morphology or the TUNEL reaction was not observed prior to blastocyst formation around the 32-cell stage in mouse in vivo and in vitro embryos [12, 26] or prior to compaction in normal developing human in vitro embryos [24]. Progress to the eight-cell stage coincides with the time of major genome activation in cattle, whereas this event occurs around the two-cell stage in mouse and human embryos [41]. The concurrence of genome activation with competence for DNA degradation in bovine in vitro embryos may be caused by deviant activation of apoptosis suppressing genes in some of these embryos [6, 42]. The present results indicate that passing this point of development under presumably optimal conditions in vivo does not result in a similar activation of the apoptotic program, suggesting a tentative relation between accelerated apoptotic activity and deviant genome activation in preimplantation bovine embryos. However, the onset of apoptosis is not observed before compaction in both mouse and human in vitro embryos, possibly because the relation between genome activation and onset of apoptosis is not as straightforward or because the IVP systems are more optimized in these species.
Fragmentation of condensed nuclei by karyorhexis is another key element of apoptosis that may be affected by IVP. This feature was not observed before the morula stage in vivo, but it seemed accelerated to the 9- to 16-cell stage in vitro. Thus, different features of apoptosis appeared at various developmental stages, they were differentially affected by IVP, and their appearance may be dependant on species-specific characteristics.
In cattle, the appearance of different apoptotic elements is not fixed, and IVP affects their occurrence. Various molecular components of the apoptotic cascade are present in early cleavage stages of mouse [43], human [44], and bovine [8, 23] embryos. These findings are supported by results from chemical induction of the TUNEL reaction at stages where apoptosis is not occurring spontaneously in both mouse [45] and bovine [6] in vitro embryos. Such results indicate that cleavage stage blastomeres constitutively process the machinery to run the apoptotic program if adequately provoked. However, whereas chemical induction may activate the full apoptotic machinery, stressors or sublethal insults may only partially activate the process in preimplantation embryos. A recent study of bovine in vitro embryos documented that heat stress can induce the TUNEL reaction at the late 8- to 16-cell stage but not at the 2- to 4-cell stage [8]; however, an effect of heat stress on subsequent cell numbers was observed in 2- to 4-cell embryos, and nuclear fragmentation was found in a heat stressed 2-cell embryo, documenting that this feature of apoptosis was affected by the treatment. Thus, the difference in effect of IVP and stress on developmental regulation of nuclear fragmentation and DNA degradation may indicate governance of these discrete apoptotic features by separate mechanisms in early embryo stages. This hypothesis is supported by results in other cell systems, where changes in nuclear morphology and DNA degradation are probably differentially regulated [32, 46]. Culture conditions that cause an increased incidence of the TUNEL reaction in preimplantation embryos [19, 47] have no significant effect on the incidence of nuclear fragmentation. If the gradual occurrence of different apoptotic features during preimplantation development reflects a progressive release of repression of these elements in the constitutively present program, then the release of this repression could possibly be individually modulated for each feature by ambient conditions such as IVP procedures and various stressors. Thus, premature occurrence of different apoptotic features may serve as indicators of stressors that may affect one but not necessarily all features of apoptosis in early embryonic stages.
The postimplantation developmental potential or embryo quality is likely to be affected by apoptotic incidence in preimplantation stages. Thus, the degree and patterns of cell fragmentation have an impact on implantation and development [48], and culture conditions that decrease embryonic cell number and increase the apoptotic incidence also decrease implantation rates, increase fetal resorption, and lower fetal birth weight upon embryo transfer [9]. A negative correlation between embryonic cell number and incidence of the TUNEL reaction has been established in both mouse [11, 26] and bovine [14] embryos, and the incidence of the TUNEL reaction is higher in mouse in vitro embryos than in similar in vivo embryos [26]. In the present study, almost every in vivo and in vitro blastocyst displayed at least one apoptotic nucleus. This finding is consistent with those of previous studies [6, 14, 27] and indicates the universal occurrence of this cellular process during normal bovine development. Although the appearance of apoptotic characteristics was developmentally accelerated by IVP, these features were only observed in insignificant numbers of precompaction embryos. Likewise, incidence of apoptosis at the morula stage was not significantly affected. These results indicate that even though IVP affects the chronological occurrence of apoptosis, a substantial impact on apoptotic incidence may not occur prior to compaction. However, a stage-specific decrease in apoptosis was previously reported in bovine in vitro morulae [14] and could explain the lack of difference in apoptotic activity at this specific stage. The higher number of cells in vivo morulae could have been generated by erroneous inclusion of blastocysts that may have collapsed during the flushing and collection procedure, thereby biasing this experimental group.
The incidence of apoptotic nuclei and nuclei displaying apoptotic features was higher in bovine in vitro blastocysts than in their in vivo counterparts. This finding is similar to that for mouse embryos [26] and supports a relation between incidence of cell death and developmental potential. Apoptotic incidence was higher in the ICM than in the Tb compartment regardless of production system, as has been observed in other studies of in vitro blastocysts from cattle [6, 22, 27], mice [11], and rats [49]. However, such a difference is not apparent in human embryos [13]. Differences observed at the blastocysts stage were specifically based on differences of cell death activity in the ICM; no differences were observed in the Tb compartment between production systems. This finding may have substantial importance, because the pluripotent ICM forms the future embryo proper, and damaging effects only affecting this embryonic compartment may result in blastocysts that appear normal at the stereo microscopical level but that carry subcellular deviations that could impact on developmental competence [50].
Apoptosis occurs during normal preimplantation development of bovine embryos produced in vivo and in vitro. By examining nuclear morphology and the incidence of the TUNEL reaction, apoptosis was identified from the 6-cell stage in vitro and the 21-cell stage in vivo, and a higher incidence of apoptotic cells was observed in blastocysts derived in vitro than in their in vivo counterparts. This difference at the blastocyst stage was specifically based on higher levels of apoptosis in the in vitro ICM.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received: 8 November 2002.
First decision: 13 December 2002.
Accepted: 21 May 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A. Jurisicova and B. M Acton Deadly decisions: the role of genes regulating programmed cell death in human preimplantation embryo development Reproduction, September 1, 2004; 128(3): 281 - 291. [Abstract] [Full Text] [PDF]
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L.A. Favetta, C. Robert, E.J. St.John, D.H. Betts, and W.A. King p66shc, but not p53, is involved in early arrest of in vitro-produced bovine embryos Mol. Hum. Reprod., June 1, 2004; 10(6): 383 - 392. [Abstract] [Full Text] [PDF]
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A. R. Beker-van Woudenberg, H. T.A. van Tol, B. A.J. Roelen, B. Colenbrander, and M. M. Bevers Estradiol and Its Membrane-Impermeable Conjugate (Estradiol-Bovine Serum Albumin) During In Vitro Maturation of Bovine Oocytes: Effects on Nuclear and Cytoplasmic Maturation, Cytoskeleton, and Embryo Quality Biol Reprod, May 1, 2004; 70(5): 1465 - 1474. [Abstract] [Full Text] [PDF]
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A Kidson, F J Rubio-Pomar, A Van Knegsel, H T A Van Tol, W Hazeleger, D W B Ducro-Steverink, B Colenbrander, S J Dieleman, and M M Bevers Quality of porcine blastocysts produced in vitro in the presence or absence of GH Reproduction, February 1, 2004; 127(2): 165 - 177. [Abstract] [Full Text] [PDF]
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H. M. Knijn, J. O. Gjorret, P. L.A.M. Vos, P. J.M. Hendriksen, B. C. van der Weijden, P. Maddox-Hyttel, and S. J. Dieleman Consequences of In Vivo Development and Subsequent Culture on Apoptosis, Cell Number, and Blastocyst Formation in Bovine Embryos Biol Reprod, October 1, 2003; 69(4): 1371 - 1378. [Abstract] [Full Text] [PDF]
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