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BOR - Papers in Press, published online ahead of print May 28, 2003.
Biol Reprod 2003, 10.1095/biolreprod.102.013243
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BIOLOGY OF REPRODUCTION 69, 1193–1200 (2003)
DOI: 10.1095/biolreprod.102.013243
© 2003 by the Society for the Study of Reproduction, Inc.

Embryo

Chronology of Apoptosis in Bovine Embryos Produced In Vivo and In Vitro

Jakob O. Gjørret1,2, Hiemke M. Knijn3, Steph J. Dieleman3, Birthe Avery4, Lars-Inge Larsson2, and Poul Maddox-Hyttel2

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The postimplantation developmental potential of embryos can be affected by various forms of cell death, such as apoptosis, at preimplantation stages. However, correct assessment of apoptosis is needed for adequate inference of the developmental significance of this process. This study is the first to investigate the independent chronological occurrence of apoptotic changes in nuclear morphology and DNA degradation (detected by the TUNEL reaction) and incidences of nuclei displaying these features at various preimplantation stages of bovine embryos produced both in vivo and in vitro. Different elements of apoptosis were observed at various developmental stages and appeared to be differentially affected by in vitro production. Nuclear condensation was observed from the 6-cell stage in vitro and the 8-cell stage in vivo, whereas the TUNEL reaction was first observed at the 6-cell stage in vitro and the 21-cell stage in vivo. Morphological signs of other forms of cell death were also observed in normally developing embryos produced both in vivo and in vitro. The onset of apoptosis seems to be developmentally regulated in a stage-specific manner, but discrete features of the apoptotic process may be differentially regulated and independently modulated by the mode of embryo production. Significant differences in indices of various apoptotic features were not evident between in vivo- and in vitro-produced embryos at the morula stage, but such differences could be observed at the blastocyst stage, where in vitro production was associated with a higher degree of apoptosis in the inner cell mass.

apoptosis, developmental biology, early development, embryo, in vitro fertilization


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Functional mechanisms for disposal of cells that are in excess, in the way, abnormal, or potentially dangerous are crucial during development and for tissue homeostasis in multicellular organisms. The phenomenon of cell death is firmly established in preimplantation development of mammalian embryos (for reviews, see [1, 2]), and roles and regulation of these processes are currently under investigation. In classic terms, necrosis and apoptosis are two types of cell death that can be differentiated by their morphological appearance [3, 4]. However, several intermediate forms exist [5], and several types of cell death may occur during normal embryonic development. Necrosis, an accidental form of cell death resulting from a direct injury, commonly affects cells in clusters and inflicts damage on their neighbors by triggering an inflammatory response, whereas apoptosis, a highly conserved process, is a far more regulated and suicidal form of cell death that occurs in single cells. Apoptosis has received an increasing amount of attention because of its potential role in early embryonic loss and in cellular responses to stress and suboptimal developmental conditions [6–11]. However, an accurate assessment of the different forms of cell death is needed to adequately infer their biological significance during preimplantation development.

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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
In Vivo Embryo Production

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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  		FIG. 1. A) Confocal laser scanning image of a 10-cell in vivo-produced bovine embryo. The chromatin content is stained by PI (red), fragmented DNA is labeled by the TUNEL reaction (green), and colocalization with PI is indicated as yellow. A highly condensed (pyknotic) but not fragmented nucleus (a) can be distinguished from normal appearing nuclei (b) with reticulated chromatin content and the condensed chromosomes organized in a metaphase plate (c). The condensed nucleus is observed in a slightly marginalized blastomere at the periphery of the cell aggregation made up by the other embryonic blastomeres; however, it is not yet displaying the TUNEL reaction. B) Confocal laser scanning images of embryonic nuclei. Note the normal appearing nuclei (d) and nuclei displaying typical morphological features of apoptosis (nuclear condensation and fragmentation by karyorhexis) without (e) or with (f) the TUNEL reaction. Nuclei and fragments thereof are all displaying sharp delineations. C) Confocal laser scanning images of embryonic nuclei. Note the nuclei displaying the TUNEL reaction and a lack of volume reduction but with an unclear or fluffy delineation (g) when compared with normal nuclei (h: two partially overlapping nuclei). D) Schematic diagram presenting the classification of nuclei according to two markers of apoptosis. Nuclei only displaying the morphological characteristic of apoptosis belong to the area termed +M (red), nuclei only displaying the biochemical characteristic of apoptosis (the TUNEL reaction) belong to the area termed +T (green), nuclei displaying both features (and fulfilling the criteria for apoptosis in this study) belong to the area termed M&T (overlap), and all nuclei displaying either one or both characteristics are defined as M+T (red and green). E and F) Confocal laser scanning images of in vivo-produced (E) and in vitro-produced (F) blastocysts. Note the presence of nuclei displaying apoptotic morphology with (i) or without (j) the TUNEL reaction and nuclei with the TUNEL reaction but no apparent nuclear condensation (k). Nuclei displaying apoptotic features are predominantly present in the ICM. G) Schematic presentation of chronological occurrence of cytoplasmic fragmentation, changes in nuclear morphology, DNA degradation, and cell marginalization at various stages of bovine preimplantation development in vivo and in vitro. Cytoplasmic fragmentation, nuclear condensation, and cell marginalization (broken line) and extrusion (solid line) occur concurrently in embryos from the two production systems, whereas nuclear fragmentation and DNA degradation can be observed at earlier stages in vitro than in vivo. Bars = 20 µm

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).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Chronological Changes in Apoptotic Morphology and DNA Degradation

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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  		TABLE 1. Numbers of embryos analyzed and occurrence and distribution of apoptotic changes in nuclear morphology and TUNEL reaction at different preimplantation stages of bovine embryos produced in vivo and in vitro. No differences in percentages were observed at any stage between in vivo- and in vitro-produced embryos (P > 0.1, {chi}2 test)

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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  		TABLE 2. Mean (±SEM) number of cell in the trophoblast (Tb), the inner cell mass (ICM), and the total blastocyst and percentage of nuclei displaying morphological changes of apoptosis (+M), TUNEL reaction (+T), both markers concurrently (M & T), and one or both (M + T) markers for bovine blastocysts produced either in vivo (n = 35) or in vitro (n = 31)

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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  		FIG. 2. Relationship between cell number and apoptotic index in the ICM of bovine blastocysts produced either in vivo or in vitro. R2a and R2b are the correlation coefficients for observations made in the ICM in vivo and in vitro, respectively


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Occurrence and percentage of nuclei displaying morphological changes compatible with apoptotic cell death and DNA degradation detectable with the TUNEL reaction were assessed in this study. Only nuclei concurrently displaying features of both apoptotic morphology and the TUNEL reaction were regarded as apoptotic. This approach gives a relatively conservative estimate of apoptotic activity; however, there are several reasons for applying both morphological and biochemical markers simultaneously in assessing apoptosis. Morphological evaluation is crucial because different cell death pathways share biochemical features [4, 5, 33]. A substantial proportion of TUNEL-positive nuclei did not display concurrent apoptotic morphology, indicating that necrosis or possibly other modes of cell death may occur in apparently normal in vivo and in vitro embryos. However, TUNEL-positive nuclei lacking apoptotic morphology may also represent blastomeres suffering the fate of secondary necrosis [33, 40], which is a normal process in apoptotic cells that are not being adequately removed by phagocytosis. Biochemical markers of apoptosis are equally important to proper assessment. Nuclei displaying apoptotic morphology but no TUNEL reaction were observed at all embryonic stages after the eight-cell stage regardless of production system. Such a lack of TUNEL reaction may have different reasons. Extensive endogenous DNA degradation is a relatively late event in the apoptotic cascade [32], and some nuclei with apoptotic morphology and no TUNEL reaction may not have reached this stage yet. Other possibilities could be misinterpretation of prophase nuclei, which may display increased staining intensity caused by higher chromatin content than that seen in interphase nuclei, or the irregular appearance of metaphase plates or telophase nuclei on confocal optical sections, making them hardly distinguishable from condensed and lobulated or fragmenting nuclei. These problems emphasize the need for adequate staining and confocal imaging procedures, even though great efforts were invested in these areas. Nuclei also may become fragmented by mechanisms independent of the apoptotic program, such as uncontrolled chromosome segregation during mitosis, which may generate micronuclei-like structures by dislodgement of chromosomes. The complex expression of morphological and biochemical markers of apoptosis highlights the need for a combination of criteria when occurrence and function of apoptosis is assessed [33]. This is the first study to investigate the independent chronological appearance of apoptotic nuclear morphology and DNA degradation during preimplantation development both in vivo and in vitro, and the results support the argument for implementing such an approach when quantification of apoptosis in embryos is desired.

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.


   ACKNOWLEDGMENTS
 
The authors thank Anne Dorthe Rasmussen for technical assistance with in vitro embryo production, Susanne Holm Kristiansen for technical assistance with the TUNEL reaction, Michael Hansen for help with confocal equipment, Jytte Nielsen for technical assistance with image processing, Bo Martin Biby for advice on statistical procedures, and technical personnel for animal care and handling.


   FOOTNOTES
 
1 Correspondence: Jakob Oemar Gjørret, Department of Anatomy and Physiology, Royal Veterinary and Agricultural University, Grønnegårdsvej 7, DK-1870 Frederiksberg C, Denmark. FAX: 45 3528 2547; jog{at}kvl.dk Back

Received: 8 November 2002.

First decision: 13 December 2002.

Accepted: 21 May 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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