1 Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309 and 2 Colorado Reproductive Endocrinology, Rose Medical Center, Denver, CO 80220, USA
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Abstract |
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Key words: `comet' assay/electron microscopy/embryo fragmentation/time-lapse video microscopy/TUNEL
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Introduction |
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Other common fragmentation phenotypes exhibited by early human embryos are the occurrence of multiple clusters of fragments, which arise during early cleavage and persist as the embryo continues to divide, or which seem to `disappear' during culture resulting in apparently normal-appearing embryos at transfer. These specific patterns of fragmentation are perhaps the most difficult to evaluate for competence because it is unclear whether they indicate an underlying defect in the early embryo that could have potentially adverse consequences during later stages of embryo development (Antczak and Van Blerkom, 1999). In clinical IVF, it can be difficult to distinguish between fragmentation that is acute or progressive if assessments of embryo development are infrequent. Here, temporal, spatial, and morphodynamic aspects of these fragmentation patterns were analysed from the pronuclear to the 12-cell stage by means of time-lapse video and TEM. Alterations in the composition of the plasma membrane and the integrity of the DNA which could signify premorbid alterations at the blastomere level were assessed by annexin V staining and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling) or single-cell alkaline gel electrophoresis (`comet') assays respectively.
The results provide new insight into the morphodynamics of fragmentation and for these embryos, demonstrate that fragments are not stationary, that many apparent fragments are actually bulbous/bleb-like elaborations of the plasma membrane and subjacent cytoplasm, rather than discrete extracellular structures, and that the disappearance of `fragments' during culture may occur by resorption or lysis. Competence for these embryos is suggested by the absence of detectable DNA damage or plasma membrane alterations and successful outcome after transfer. The absence of mitochondria in most of the fragments detected in these embryos is discussed with respect to a non-apoptotic aetiology.
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Materials and methods |
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Images were captured on an optical disc recorder at intervals ranging from 30 s to 10 min, depending upon the stage of development. For some analyses, embryo recordings were made under constant, low-light illumination using image intensification afforded by high-resolution video cameras. In these instances, simultaneous optical disc (images captured at 5 min) and videotape recordings (images captured at 1 s intervals, with a time-lapse videocam recorder) were made in order to time precisely the occurrence of specific morphodynamic events. Protocols for time-lapse recording and standard procedures for electron microscopy and analysis of serial thin sections have been described previously (Van Blerkom et al., 1995
).
Detection of DNA breaks
TUNEL analysis
Fragmented embryos with intact blastomeres were prepared for TUNEL analysis by two different methods: as whole mounts (Takase et al., 1995; Van Blerkom and Davis, 1998
), or as air-dried preparations on microscope slides (Jurisicova et al., 1996
; Perez et al., 1999
). Both procedures used reageants supplied in an In Situ Cell Detection Kit with fluorescein-tagged nucleotides (Roche Molecular Biochemicals, Indianapolis, IN, USA). TUNEL analysis used zona-intact, zona-thinned, and zona-denuded specimens (Antczak and Van Blerkom, 1999
). Briefly, samples were fixed in a freshly prepared 3.7% formaldehyde solution in phosphate-buffered saline (PBS, pH 7.35) for 45 min and washed for >12 h in PBS. Zona-free embryos were washed in PBS containing 4% poyvinylpyrrolidone. Samples were incubated in TUNEL reagents for 1 h at 37°C according to the recommendations of the manufacturer and the findings of our previous studies. Embryos were treated with Slow-Fade Lite (Molecular Probes, Eugene, OR, USA) and examined first by conventional epifluorescence microscopy with ultraviolet illumination and then by scanning laser confocal microscopy (SLCM) to obtain serial optical sections through entire specimens. All TUNEL-negative specimens were treated with DNase II (Sigma Chemical Co., St Louis, MO, USA) and restained with TUNEL reageants as described previously (Van Blerkom and Davis, 1998
).
For air-dried samples, embryo preparation for TUNEL involved partial zona thinning with acidic Tyrode's (1525 s) in order to retain fragments. After denudation, specimens were immediately fixed for 3045 min in a freshly prepared 3.7% formaldehyde solution (pH 7.2) during which the zona remnant was removed by microneedle dissection. Fixed embryos were washed in PBS, deposited by micropipette in 2 µl of fluid onto treated slides (Superfrost Plus; Fisher Scientific, Pittsburgh, PA, USA) and air-dried. Prior to deposition, a 10 mm square box was delineated with a Pap Pen (Sigma Chem. Co.) in order to confine solutions and reageants to a specific location. Slides were incubated at 65°C for 4h, stored overnight at 4°C, reheated for 30 min at 65°C, then rehydrated through a graded series of ethanol, and gently washed in multiple changes of high-purity, cell culture-grade water. Slides were incubated for 30 min at 37°C in PBS containing proteinase K at a concentration of 10 µg/ml (Roche Molecular Biochemicals). After enzymatic treatment, slides were washed in PBS containing 3% bovine serum albumin (BSA) at room temperature, excess fluid was withdrawn by micropipette, and the samples immediately exposed to TUNEL reagents as described above. TUNEL staining was performed at 37°C for 1.5 h followed by numerous rinses in PBS. Slides were placed in Slow Fade Lite equilibration buffer for 30 min (Molecular Probes, Eugene, OR, USA), excess buffer removed with a micropipette, and a small drop of Slow Fade was placed over the specimen and covered with a glass coverslip. Specimens were examined by conventional epifluorescence microscopy and by SLCM. After examination, the coverslip was gently removed and the slides washed in HEPES-buffered saline (pH 7.4), stained with DAPI for 15 min, and rinsed in HEPES-buffered saline for 30 min. Excess buffer was removed and replaced with 70% glycerol, and the specimens re-examined to detect chromosomal and nuclear DNA. All TUNEL-negative specimens were rehydrated, washed in PBS, and exposed to either DNase or to PBS containing 0.5 mol/l H2O2 for 15-30 min. These slides were processed and restained with TUNEL reagents as described above.
`Comet' analysis
The following describes in detail the standard protocol we devised for single cell alkaline gel electrophoresis (`comet' assay) which was modified from Singh and Stephens (1997). After partial thinning of the zona pellucida, embryos were washed in HEPES-buffered human tubal fluid medium (Quinns's HTF) and deposited onto a treated microscope slide in a 2 µl volume (Comet Slides, Trevigen, Inc., Gaithersburg, MD, USA). Most of the fluid surrounding the specimen was carefully and rapidly removed by a mouth-operated micropipette drawn to a fine taper. A 3 µl drop of a 0.4% solution of agarose (high-resolution blend 3:1 agarose; Amrecso, Solon, OH, USA) prepared in calcium- and magnesium-free PBS (pH 7.4) kept at 43°C was deposited over the embryos and gently extended outward with a micropipette whose tip had been fire-polished to form a small bead. Glass microneedles were used to disrupt mechanically the remnant zona, allowing blastomeres and fragments to be completely encapsulated by agarose within a confined area. The residual zona had no effect on the `comet' assay. After solidification, the agarose layer was immediately surrounded by a 25 µl ring of agarose kept at 45°C. With a beaded micropipette, the molten agarose was rapidly extended inward to contact the agrose-entrapped specimen, and when joined, formed a uniformly thin agarose microgel. During the above operations, slides were observed at x20 magnification on a Nikon Diaphot inverted microscope maintained at 39°C by means of a heated chamber attached to the microscope (Nikon, NP-2 incubated chamber). The entire process, from the initial deposition of the specimen to formation of the final agarose thin layer, was performed in <60 s.
After formation of microgels, the following steps were done under reduced ambient illumination or in complete darkness: agrose microgels were placed at 4°C on a refrigerated cooling block (Millipore, Bedford, MA, USA) for 10 min and then immersed for 45 min in a lysis solution (2.5 mol/l NaCl, 100 mmol/l disodium EDTA, 10 mmol/l Tris base, 1% sodium lauryl sarcosinate, 0.1% Triton X-100, pH 10) maintained at 4°C in glass Petri dishes on the same cooled platform. For some preparations, microgels were treated with proteinase K (1 mg/ml) in detergent-free lysis solution for up to 2 h at 37°C, and then immersed in 100 ml of alkali buffer (300 mmol/l NaOH, 0.1% 8-hydroxyquinoline, 2% dimethylsulphoxide, 100 mmol/l EDTA, pH 10) for 1 h at room temperature. Microgels were immersed in TBE (Tris-borate-EDTA buffer) for 5 min and positioned in a horizontal slab gel electrophoresis apparatus, equidistant from the electrodes. Electrophoresis was performed in total darkness for exactly 12 min at 200 mA in 1 litre of pre-chilled TBE. After electrophoresis, slides were neutralized in 100 ml of 0.4 mol/l Tris, pH 7.4, excess fluid drained, and the microgels stained with SYBR Green (Molecular Probes), over-layered with a coverslip, and examined immediately by conventional epifluorescence in the fluorescein isothiocynate (FITC) channel. Slides were dehydrated in ethanol, air-dried and stored in desiccant-containing chambers at 5°C.
Annexin V staining and exposure of embryos to hypotonic solutions or hydrogen peroxide
The integrity of the plasma membrane was assessed in living specimens by the presence or absence of cytoplasmic and nuclear fluorescence after staining with propidium iodide (Van Blerkom and Davis, 1998), or by cytoplasmic incorporation of Trypan Blue (0.2% in culture medium, 5 min exposure). Phosphatidylserine translocation from the inner to outer leaflet of the plasma membrane was determined by staining living embryos with FITC-tagged annexin V (AV; Clontech, Palo Alto, CA, USA) as previously described (Van Blerkom and Davis, 1998
). Controls for the specificity of the AV, TUNEL and `comet' assays used unfragmented dispermic embryos exposed to medium containing hydrogen peroxide (500 µmol/l, 30 min) or to hypotonic conditions (distilled water or a 0.5% sodium citrate), treatments known to cause rapid loss of plasma membrane and DNA integrity by cell lysis and oxidation, respectively. Additional controls included normal 8-12-cell cryopreserved embryos that showed lethal cellular damage and lysis after thawing and rehydration.
Fluorescent microscopic determinations of mitochondrial distributions
Embryos where the occurrence of fragments could be timed by time-lapse microscopy during the pronuclear and early cleavage stages were stained with one of the following mitochondria-specific fluorescent probes (Molecular Probes) as described previously (Van Blerkom et al., 1995, 1998
): nonyl-acridine orange (NAO), rhodamine 123 (R123). The distribution of mitochondria between fragments was determined by scanning laser confocal microscopy (SLCM) analysis of living embryos, after which embryos were returned to culture.
Statistical analysis
2-analysis was used to determine whether differences in clinical pregnancy rates were significant between patients in whom transfers involved only embryos which remained intact during culture, or only embryos which exhibited the fragmentation patterns and behaviours which were studied.
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Results |
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Fragment formation at the pronuclear stage
Figure 1AH show a representative pattern of early fragmentation observed in monospermic (Figure 1A
, n = 26) pronuclear oocytes that, on day 3, appeared intact and normal. In these instances, fragment formation was detected at syngamy (asterisks, Figure 1B
) and during the first cell division (Figure 1CE
). For the embryo shown, images were captured at 1 min intervals over a 20 h period from the pronuclear (Figure 1A
) to the 4-cell stage (Figure 1H
, 39 h). However, by the 4-cell stage, virtually all fragments had disappeared resulting in a morphologically normal classification. Eighty-seven per cent (23/26) of these embryos progressed to the 12-cell stage by day 3.5. The apparent disappearance of clusters of `fragments' was a common feature of these embryos. For example, the cluster of fragments indicated by an asterisk in Figure 1G
appeared during a 15 min interval between 33 h 56 min and 34 h 11 min, but was undetectable at 34 h 26 min. After the 4-cell stage (Figure 1H
), fragments were undetectable by light microscopy on either the exposed surface or the interior of the embryo (as observed in optical sections) as cleavage progressed.
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Apparent fragment resorption and lysis
The rapidity with which fragments seemed to disappear in time-lapse images (within minutes) suggested that fragment loss may involve lysis or resorption. For cleavage stage embryos, evidence for lysis of small apical fragments (e.g. arrows, Figure 1J, M) which seemed to burst during intervals of image-capture was suggestive but not definitive. In contrast, clear evidence for lysis was obtained with larger fragments. For example, fragments (e.g. white arrows, Figure 1U, V
; white asterisks, Figure 1WY
) which began to swell and display a transparent cytoplasm seemed to disappear in a `burst-like' fashion. Membranous remnants of these fragments were detectable in the adjacent perivitelline space (arrows, Figure 1W, X
). The arrows in Figure 1Y
indicate the portion of the fragment from which cytoplasm was observed in the process of being discharged. During a subsequent 12 h culture period, other fragments in this representative embryo underwent swelling and apparent lysis. Time-lapse images provided suggestive evidence for fragment resorption, as these entities appeared to merge with the plasma membrane. A possible cellular basis for resorption was indicated by TEM findings described below.
Transmission electron microscopy
Based on the timing and behaviour of fragments observed by time-lapse, the following types of embryos (as shown in Figure 1) were examined by TEM: (i) monospermic embryos which appeared to contain `pseudo-fragments' (n = 8), (ii) monospermic embryos in which fragment lysis was suggested by changes in cytoplasmic texture and swelling (n = 9), and (iii) monospermic (n = 6) and dispermic (n = 36) embryos in which fragments detected during early cleavage were largely undetectable at the 8-12-cell stage.
The TEM images of fragmented embryos shown in Figures 2 and 3A were selected because they demonstrate fine structural characteristics common to all embryos which were examined. Spherical elements that occurred in columns were the most common and consistent type of fragment observed at the pronuclear and early cleavage stages. Typically, these structures exhibited a uniform cytoplasmic texture with virtually no organelles. Other fragments appeared lysed (L, Figure 2B, E
) or were likely candidates for lysis (i.e. prelytic, Lp, Figure 3A
). All lysed fragments were enclosed by a discontinuous plasma membrane (arrows, Figure 2F
). Figure 2A
is derived from an embryo similar to the one presented in Figure 1C
, and shows fine structural characteristics of a typical cluster of fragments that arose during the pronuclear stage. Analysis of serial sections indicated virtually no mitochondria or other cytoplasmic components present in these structures. When organelles were present in columns of fragments, they were mostly confined to the fragment directly associated with the blastomere plasma membrane, such as fragment 3 in Figure 2D
. Cytoplasmic continuity between fragments was another consistent TEM finding. In the selected images shown in Figures 2A
(pronuclear stage), 2B (4-cell), 2D, (2-cell), 2E (8-cell) and 3A (12-cell), serial section analysis demonstrated cytoplasmic continuity between fragments (e.g. 13 in Figure 2A, D
), as well as between columns of interconnected fragments and the underlying blastomere (e.g. fragment 3, Figure 2D
; asterisks, Figure 2E
; arrows, Figure 3A
). For example, the two images shown in Figure 2C
correspond to the region indicated by an arrow in the fragment labelled with a black asterisk in Figure 2B
. Cytoplasmic continuity between columns of fragments and the underlying blastomere(s) observed by serial section analysis is also indicated by asterisks in Figure 2E
and by a series of arrows in Figure 3A
. Such complexes and columns of interconnected fragments may represent `pseudo-fragments' and continuity with the underlying cell could provide a basis for resorption. The occurrence of detached fragments, including those relatively few which contained cytoplasmic components such as mitochondria (M, Figure 2E, 3A
), may result from breakage of cytoplasmic bridges during blastomere movement and rotation.
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Serial section reconstructions of late cleavage-stage embryos indicated that associations between intact fragments involved membrane specializations (large arrow Figure 2E, and at higher magnification in 2G
) similar in appearance to the occluding junctions between blastomeres in the same embryo(s). For fragments, this type of tight association could provide a mechanism for coordinated movement with an underlying blastomere(s) as detected by time-lapse or their apparent disappearance from apical surfaces if internalized, as indicated by TEM.
Analysis of plasma membrane and DNA integrity
Annexin V staining
Prior to fixation for TEM, TUNEL or `comet' analysis, embryos (n = 23) were stained with AV where time-lapse indicated (i) disappearance of fragments by the 812-cell stage (e.g. Figures 1P, 3B), or (ii) where fragments persisted through cleavage in otherwise stage-appropriate embryos (e.g. Figure 1T
; arrow, 3D
). None of these embryos exhibited blastomere-associated AV fluorescence on days 3 or 3.5 (Figure 3C
). In contrast, where fragments that first appeared at the 2-cell stage were still evident during subsequent culture, AV fluorescence was detected in some fragments. The arrow in Figure 3D
indicates a cluster of fragments in a 6-cell embryo (DAPI epifluorescent image showing four nuclei, Figure 3E
) that first appeared at the 2-cell stage. One fragment in this cluster was clearly AV positive (arrow, Figure 3F
). As a control for specificity, AV-negative embryos and embryos with an occasional positive fragment (arrow, Figure 3G
) were restained after fixation in formaldehyde, or exposure to either hydrogen peroxide or hypotonic conditions. In all cases, AV fluorescence was associated with the plasma membrane of blastomeres and retained fragments. The epifluorescent image shown in Figure 3H
is the same embryo as in Figure 3G
after fixation and restaining with AV.
TUNEL assay
TUNEL analysis was performed on 41 stage-appropriate (812-cell) day 3.03.5 embryos where fragment clusters that arose during early cleavage were either no longer apparent (n = 14) or were still detectable to different extents (n = 27). Prior to TUNEL analysis, embryos were stained with Trypan Blue to confirm blastomere integrity. Embryos were prepared either as whole mounts (n = 19) or as air-dried specimens (n = 22). Equivalent sensitivity and specificity of the two methods was confirmed in other studies that used intact and zona-free unfertilized oocytes and dispermic embryos (J.Van Blerkom, unpublished observations). For example, the epifluorescent image of an unpenetrated oocyte prepared as a whole mount in Figure 3I shows TUNEL fluorescence localized to the first polar body. The oocyte shown in Figure 3J
was an air-dried preparation that exhibited a TUNEL-positive polar body. After treatment with DNase, metaphase II chromosomal TUNEL fluorescence was detected in all oocytes (e.g. Figure 3K
). None of the embryos prepared as whole mounts (Figure 3L, M
) or air-dried (Figure 3N, O
) showed nuclear TUNEL staining, and after DNase treatment, all nuclei were TUNEL positive (Figure 3P
, the same embryo as in O; Figure 3L
is the same embryo shown in Figure 1T
). If present, the only TUNEL fluorescence detected in embryos was associated with the second polar body (arrow, Figure 3O
).
As an additional control for the specificity of this assay, unfragmented 28-cell dispermic embryos (n = 12) were exposed to hypotonic conditions and fixed for TUNEL analysis at specific times after blastomere swelling and Trypan Blue incorporation were detected. Figure 3QR are representative images of a 4-cell dispermic embryo with mononucleated blastomeres as it appeared prior to (Figure 3Q
) and after 12 min (Figure 3S
) in hypotonic conditions (Figure 3R
). Nuclear TUNEL fluorescence in this embryo as observed in a fully compiled SLCM image is shown in Figure 3S
.
`Comet' assay
For `comet' analysis, stage-appropriate embryos (monospermic, n = 6; dispermic, n = 17) were examined in which fragments that occurred during the 24-cell stage were still detectable at the 812-cell stage (e.g. Figure 1QT). The analysis was confined to these embryos because, in current clinical practice, the presence of fragments of this type could be considered indicative of reduced competence. Eighty-two per cent (172/207) of the blastomeres in 23 cleavage stage embryos prepared for `comet' were detected in the agarose minigels. Under the conditions and duration of electrophoresis used, none of the 172 nuclei displayed tails indicative of DNA fragmentation. A representative image of three fluorescent nuclei from the 8-cell embryo shown in Figure 4A
is presented in Figure 4B
. In contrast, `comet' tails (Figure 4C
) were observed in all second polar bodies that were Trypan Blue positive in the living state (arrow Figure 4A
) and could be identified in the agarose minigels prior to electrophoresis owing to significant dye incorporation.
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Mitochondrial content of fragments
Eighteen embryos (four monospermic, 14 dispermic) examined by time-lapse developed fragment clusters at the pronuclear or early cleavage stages and were apparently fragment-free or contained apical clusters at the 812-cell stage as described above. Embryos were stained with R123 within 4 h after fragments were initially detected at the pronuclear or 2-cell stage (e.g. Figure 1Q), and after SLCM analysis, were returned to culture and time-lapse observations continued. The pronuclear oocyte shown in Figure 4L
is presented because of the apparent severity of fragmentation observed at the light microscopic level. However, the fully-compiled SLCM R123-fluorescent image of this embryo (Figure 4M
) is representative of all 18 embryos that developed fragments during early development but which appeared largely normal on day 3 or 3.5. The absence of detectable mitochondrial fluorescence was characteristic of most of the fragments in these embryos (asterisks, Figure 4M
). The relatively few fragments with detectable mitochondrial fluorescence are indicated by an arrow in Figure 4M
. After SLCM examination, time-lapse analysis showed fragment resorption or lysis as described above, and if present during later development, only small clusters of fragments remained. For example, the embryo presented in Figure 4N
(day 2) is the same embryo shown in Figure 4L
and Figure 4O
is a fully-compiled SLCM image after staining with NAO. The absence of fluorescence in most of the residual fragments (arrow, Figure 4N
) was a consistent finding for progressive embryos which exhibited this pattern of fragmentation during early development.
Outcome after embryo transfer
In parallel with time lapse and biochemical analyses, the timing and pattern of fragmentation was systematically assessed in embryos destined for transfer or cryopreservation, by sequential observations (48 h) of individual embryos during 3 days of culture. Seventy-two day 3 embryos transfers to women between the ages of 28 and 37 years involved two 810-cell embryos with the pattern of fragmentation shown in Figure 1: (i) fragment clusters present during early cleavage were no longer detectable at transfer (type 1, n = 23) and (ii) some of the fragments that arose at the pronuclear or early cleavage stages were still detectable at transfer (type 2, n = 49). The clinical pregnancy rate for patients with type 1 and type 2 embryos was 61% (14/23) and 55% (27/49) respectively. Clinical pregnancy rates for a comparable patient population (n = 75) after the transfer of two normal-appearing (unfragmented during culture) 810-cell embryos was 67% (50/75). Although embryos that exhibited these two types of fragmentation patterns appeared to have an implantation rate lower than their unfragmented counterparts, these differences were not statistically significant.
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Discussion |
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Here, analytical findings and outcome results are reported with embryos that exhibited certain patterns of fragmentation not associated with early cleavage arrest. We focused on these specific phenotypes because they are both common in clinical IVF and their occurrence can be problematic in the assessment of competence. Time-lapse imaging from the pronuclear to the 1012-cell stage provided new insights into the fragmentation phenomenon that cannot be obtained or appreciated when embryos are examined infrequently. Within cohorts, fragments formed in some embryos during the pronuclear or early cleavage stages but were undetectable at later stages, resulting in morphologically normal, stage-appropriate embryos. For other embryos, some of the fragment clusters that had occurred during early cleavage were still detectable on apical surfaces of blastomeres during late cleavage. Time-lapse images indicated that fragments seemed to be resorbed into the underlying blastomere(s), while others disappeared abruptly, leaving behind membranous ghosts and debris. Often, these dynamic activities occurred in the same embryo.
Static TEM images of dynamic activities in embryos offer a possible explanation for apparent fragment resorption and lysis observed in the living state. In the types of embryos that were examined, TEM showed that fragments often occurred in columns, a finding that confirms an arrangement we have previously observed by SLCM (Antczak and Van Blerkom, 1999). The cytoplasm between fragments in columns and clusters, and between these fragments and the underlying blastomere(s), was often continuous, suggesting a means by which resorption could occur. The apparent absence of mitochondria in most detached fragments could lead to lysis, if depleted ATP concentrations are not replenished (see below). The apparent movement of fragments was a common and consistent activity observed by time-lapse with clusters of fragments changing location in concert with the movement of the underlying cell. Fragments at the apical surface of one or more blastomeres moved inward as cell(s) rotated giving the embryo a largely fragment-free appearance. During cleavage, fragments seemed to reappear in some of these embryos. Although it was not possible to determine definitively whether fragments internalized at one time were the same ones that appeared somewhat later, analysis of time-lapse images, which included notations of number, size and relative position, strongly suggested the same cohorts re-emerged into view if the fragment-containing region of the affected blastomere(s) returned to an apical location. TEM images indicating that clusters of fragments were tightly associated by occluding-type junctions provide a possible structural basis for movement in concert with the corresponding blastomere(s).
The fine structural images of 812-cell embryos were selected because they showed intact fragments, lysed fragments, and likely candidates for lysis (termed prelytic) interposed between adjacent blastomeres. It has been suggested that if positioned between blastomeres, fragments could perturb the normal close association of opposed blastomere plasma membranes required for compaction, such that their mechanical extraction during early cleavage could correct potential defects in morulation and improve outcome after transfer (Alikani et al., 1999). Routine light microscopy could not determine whether the types of fragments we observed by TEM were present between blastomeres at the 812-cell stage. Consequently, it is unknown whether, if present in some embryos, they could represent an irreversible, focal barrier to compaction. One experimental approach to this question currently under investigation involves labelling fragments from one embryo with permanent fluorescent probes followed by deposition in intact recipient (dispermic) embryos and tracking of the donated fragments by sequential SLCM observation. However, intact embryos and embryos which exhibited the fragmentation patterns reported here had comparable outcomes. This finding indicates that the occurrence of these fragmentation phenotypes during early development is not necessarily developmentally lethal.
Activation of programmed cell death (PCD) pathways leading to blastomere apoptosis has been suggested to be a proximate cause of fragmentation in cleavage stage human embryos, especially when significant fragmentation is accompanied by cleavage arrest (Jurisicova et al., 1996) or is so extensive that no intact blastomeres are detectable (Yang et al., 1998
). For intact 10-cell embryos, the detection of intense AV staining of the plasma membranes suggested to Levy et al. (1998) that early processes associated with PCD may be initiated in normal-appearing embryos. Therefore, even if the embryos described here appear normal on day 3, the possibility remains that lethal alterations in plasma membrane composition and DNA structure that could contribute to subsequent developmental failure may affect some blastomeres. The clinical relevance of this possibility is indicated by the detection of TUNEL-positive nuclei indicative of PCD and apoptosis (Hardy, 1999
) in cultured morula and blastocyst stage human embryos. Here, day 3 and day 3.5 embryos were examined in which fragment clusters were classified as transient or persistent for evidence of impending cell death indicated by changes at the plasma membrane (AV staining) and DNA concentrations (TUNEL and `comet' assays).
The TUNEL assay is a widely used histochemical marker that detects DNA breakage associated with apoptosis. However, TUNEL staining does not reliably discriminate between cell death that results from necrosis, autolysis and apoptosis (Kressel and Groscurth, 1994; Charriaut-Marlangue and Ben-Ari, 1995; Grasl-Kraupp et al., 1995
; Frankfurt et al., 1996
). In addition, normal nuclei undergoing active transcription can also be TUNEL positive (Kockx et al., 1998
). Consequently, the `comet' assay was also used, a method reported to be capable of identifying patterns of early DNA cleavage associated with different aetiologies of cell death. The `comet' assay is a single cell analysis of DNA integrity that is based upon the ability of denatured, cleaved DNA to migrate from the nucleus in an agrose gel when an electric current is applied. After electrophoresis, DNA is resolved with fluorescent stains and, if compromised, the fluorescent patterns resemble a comet, where tail characteristics such as shape and length can be used to determine the type of cell death (necrosis, lysis, apoptosis) and extent of DNA damage (Singh et al., 1988
; Singh and Stephen, 1997). In contrast, intact, supercoiled DNA remains within the confines of the nucleus and appears as a highly fluorescent `head' with no detectable tail. The `comet' assay has been recently used to detect DNA cleavage induced in cultured hamster embryos by hydrogen peroxide treatment, ultraviolet irradiation, or prolonged exposure to visible light (Takahashi et al., 1999
). Translocation or `membrane flipping' of phosphatidylserine (PS) from the inner to outer leaflets of the plasma membrane is an early and apparently characteristic indication of apoptosis (Martin et al., 1995
). Externalization of this moiety can be identified after staining living cells with tagged AV. However, owing to loss of plasma membrane integrity, cells undergoing necrosis or lysis will also stain positively because they permit entrance of AV and labelling of PS on the inner leaflet of the plasma membrane. Collectively, these analytical methods should detect subtle changes in plasma membrane and DNA integrity in the classes of human embryos that were examined.
The results of the current study show no indication of nuclear DNA cleavage detectable by `comet' and TUNEL assays, or loss of plasma membrane integrity detectable by AV staining. TUNEL-negative embryos were all positive after DNase treatment, and both positive TUNEL fluorescence and `comets' were evident in second polar bodies, and in all embryos where DNA cleavage occurred as a consequence of cell lysis or was induced experimentally. Embryos, that were AV negative in the living state were all AV positive after fixation or exposure to hydrogen peroxide or hypotonic conditions. This may explain the intense plasma membrane staining observed by Levy et al. (1998) for intact 10-cell embryos, which were AV stained after fixation. AV-positive structures detected in the present study may be lysed fragments with a discontinuous plasma membrane, as indicated in TEM images.
The absence of detectable alterations at the plasma membrane and DNA concentrations, combined with time-lapse, TEM findings and outcome, indicate that the patterns of fragmentation described here are not characteristic of apoptosis or indicative of PCD. While the underlying cause(s) of the fragmentation patterns described here are unknown, it is intriguing that these activities resemble some of the events associated with another form of cell death, oncosis. The characteristics of oncosis in general and how they differ from apoptosis in particular have been described in detail by Majno and Joris (1995). When deprived of oxygen (ischaemic conditions), certain cell types elaborate plasma membrane blebs devoid of organelles. Over a period of hours, these blebs progressively swell by fluid uptake as unreplenished ATP reserves diminish with increased permeability resulting from failure of plasma membrane-associated ionic pumps. At the terminal stage of oncosis, DNA fragmentation occurs and when fluid accumulation reaches lethal amounts, the affected cell undergoes lysis. As observed in culture, oncotic blebs are continuous evaginations of the plasma membrane and, if they become detached from the affected cell, they `float away' (Majno and Joris, 1995). However, if normal conditions are restored sufficiently early, resorption of blebs and return of cell function and viability can occur.
Correlated time-lapse and electron microscopic analyses provide a possible aetiology for these fragmentation patterns and for the absence of adverse effects on competence. Most of the intact fragments examined by serial thin section reconstruction contained few, if any, detectable mitochondria. This observation was confirmed in living embryos with fluorescent probes that detected active mitochondria, or all mitochondria, regardless of activity, although some fluorescent fragments were always observed. It is suggested that the longevity of detached fragments may be related to mitochondrial complement and their relative contribution to ATP concentrations by oxidative phosphorylation. Swelling and lysis may result if unreplenished ATP stores decline below a critical threshold required to maintain plasma membrane ionic pumps and cytoplasmic integrity.
The fragmentation patterns observed by time-lapse microscopy and the TEM findings showed swelling of detached fragments prior to lysis and the occurrence of columns and clusters of organelle-free elaborations of the plasma membrane in which cytoplasmic continuity with the affected cell persisted. Taken together, these features seem to resemble the initial stages of an oncotic-like process. However, an oncotic-like process would seem to be an unlikely explanation for the fragmentation phenotypes described here because culture does not occur under conditions of severe oxygen deprivation and not all embryos are affected. One possible association with an ATP-driven oncosis-like process may be related to mitochondrial distributions in pronuclear oocytes and early blastomeres, where relatively large regions of cortical cytoplasm can be permanently or transiently devoid of mitochondria (Van Blerkom, 2000; Van Blerkom et al., 2000
). For example, disproportionate mitochondrial segregation at the first cleavage division can result in some human blastomeres where the inherited complement of these organelles may be unable to contribute ATP at concentrations sufficient to maintain normal cell function and integrity in cortical regions (Van Blerkom et al., 2000
). For the fragmentation patterns described, we are investigating whether mitochondria-depleted regions of pronuclear oocytes and blastomeres exist, and whether they experience a corresponding depletion of ATP that could have focal effects on plasma membrane function associated with the elaboration of mitochondria-free elements similar to nascent oncotic blebs. According to this notion, restoration of an appropriate mitochondrial density could result in resorption where cytoplasmic continuity with the underlying blastomere was maintained, or lysis of detached structures when ATP concentrations can no longer maintain membrane integrity.
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Submitted on August 10, 2000; accepted on January 15, 2001.