Molecular Human Reproduction, Vol. 5, No. 5, 414-420,
May 1999
© 1999 European Society of Human Reproduction and Embryology
Fragmentation and death (a.k.a. apoptosis) of ovulated oocytes
Vincent Center for Reproductive Biology, Department of Obstetrics and Gynecology, Massachusetts General Hospital/Harvard Medical School, 55 Fruit Street, Boston, MA 02114, USA
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Accumulating evidence indicates that fragmentation of ovulated murine oocytes, resulting spontaneously or following exposure to lethal stimuli such as anticancer drugs during in-vitro culture, occurs with several hallmark features of apoptosis. However, recent work has failed to demonstrate a correlation between DNA cleavage, as assessed by DNA 3'-end-labelling, or of phosphatidylserine exposure on the outer leaflet of the plasma membrane, as measured by annexin V-staining, with fragmentation of ovulated mouse or human oocytes maintained in vitro. Consequently, these authors stated that it is `premature to conclude that apoptosis occurs in ovulated oocytes or that such a mechanism is involved in the elimination or prevention of fertilization of oocytes with cytoplasmic or chromosomal defects'. Here, we have re-assessed DNA cleavage in normal and fragmented murine oocytes, have provided new evidence of an additional biochemical marker of apoptosis in fragmented oocytes (i.e. caspase activity), and have re-evaluated published reports regarding oocyte fragmentation, in an effort to clarify these discrepant findings. The results and discussions presented herein fully support previous conclusions reached by ourselves and others that fragmentation of ovulated oocytes is in fact an unequivocal example of apoptotic cell death.
apoptosis/oocyte/DNA cleavage/caspase/cell death
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Introduction |
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A landmark series of morphological studies (Kerr et al., 1972
), on the events underlying normal cell turnover in the body, led to the coining of the term `apoptosis' to describe a form of cell death, clearly distinct from primary necrosis, that serves to balance mitosis in the regulation of appropriate cell numbers in various tissues (Kerr et al., 1972, 1994). The primary features of a cell undergoing apoptosis were described as `nuclear and cytoplasmic condensation and breaking up of the cell into a number of membrane-bound, ultrastructurally well-preserved fragments', subsequently referred to as apoptotic bodies. Kerr et al. (Kerr et al., 1972) went on to state that `the formation of apoptotic bodies involves marked condensation of both nucleus and cytoplasm, nuclear fragmentation, and separation of protuberances that form on the cell surface to produce many membrane-bounded, compact, but otherwise well-preserved cell remnants of greatly varying size'. Since this report, many biochemical and molecular markers of apoptotic cells have been identified, including DNA cleavage analysed by either biochemical (Wyllie, 1980) or in-situ (Gavrieli et al., 1992) approaches (detailed and reviewed in Tilly, 1994), caspase-mediated proteolysis of specific intracellular proteins (e.g. poly(ADP-ribose) polymerase (PARP), reviewed in Rosen and Casciola-Rosen, 1997; Duriez and Shah, 1997), and `flipping' of phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane (commonly detected by, and thus referred to as, annexin V-staining; see van Engeland et al., 1998). While these latter biochemical markers have certainly proved useful for detection of apoptosis, the gold standard for assessing the occurrence of apoptosis remains the morphological criteria detailed 27 years ago (Kerr et al., 1972) (for review see Kerr et al., 1994; Hockenberry, 1995; Majno and Joris, 1995).
In the past decade, tremendous progress has been made in understanding the role of apoptosis in normal tissue function as well as in the development of pathological or disease states (for review, see Thompson, 1995). Moreover, a number of genes have been identified that appear to function in an evolutionarily-conserved programme of cell death precisely orchestrated by the actions and interactions of the cell death-regulatory proteins encoded by these genes (for review see Steller, 1995; Wyllie, 1996; Yang and Korsmeyer, 1996; Golstein, 1997). Like most other major organ systems, the female reproductive tract has recently received considerable attention as a research model for cell death evaluations, with the gonad probably being the most well-studied in this context (for review see Tilly, 1998). Over the past few years, however, apoptosis research as it relates to ovarian function has begun to shift from an in-depth analysis of granulosa cell demise during follicular atresia towards a greater understanding of the possible role of apoptosis in female germ cell degeneration.
Although apoptosis, as assessed by a wide variety of criteria, is now accepted as the mechanism underlying prenatal attrition of oogonia and oocytes in the developing fetal ovary (Coucouvanis et al., 1993; Ratts et al., 1995; De Pol et al., 1997; Morita et al., 1999a) and oocyte death during primordial and primary follicle atresia in the post-natal ovary (Perez et al., 1999), one of the first reports of apoptosis in ovulated oocytes (Takase et al., 1995) concluded that apoptosis, as defined by both morphological criteria and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) analysis of DNA cleavage, is the mechanism responsible for degeneration of unfertilized murine oocytes maintained in vitro. The following year, Fujino et al. (Fujino et al., 1996), while studying in mice the relationship between maternal age and rates of spontaneous oocyte fragmentation in vitro, concurred with the work of Takase et al. (Takase et al., 1995) that apoptosis is responsible for fragmentation of murine oocytes cultured in vitro. This study was then shortly followed by two reports from our laboratory, one of which confirmed and extended the work of Fujino et al. (Fujino et al., 1996) on spontaneous fragmentation of oocytes harvested from young and aged female mice following in vitro culture (Perez and Tilly, 1997). The second study from our group assessed the occurrence and regulation of fragmentation of ovulated murine oocytes cultured in vitro without and with the anticancer drug, doxorubicin (Perez et al., 1997). In all four studies cited above, and in a fifth study recently published on the role of a specific pro-apoptotic gene product (i.e. caspase-2) in mediating both normal oocyte attrition in vivo and anti-cancer drug-induced oocyte fragmentation in vitro (Bergeron et al., 1998), the overall conclusion that oocyte fragmentation is the result of apoptosis was deduced from both morphological assessments as well as biochemical analyses of DNA integrity.
A recent investigation (Van Blerkom and Davis, 1998) on the molecular basis of oocyte fragmentation in vitro has, however, challenged the conclusion that ovulated oocytes die in vitro through the process of apoptosis. Using TUNEL and annexin V-staining, these authors failed to demonstrate a correlation between DNA cleavage or PS exposure with fragmentation of murine or human oocytes cultured in vitro (Van Blerkom and Davis, 1998). From these findings, it was argued by Van Blerkom and Davis (Van Blerkom and Davis 1998) that it is `premature to conclude that apoptosis occurs in ovulated oocytes or that such a mechanism is involved in the elimination or prevention of fertilization of oocytes with cytoplasmic or chromosomal defects.' Since we (Perez and Tilly, 1997; Perez et al., 1997; Bergeron et al., 1998) and others (Takase et al., 1995; Fujino et al., 1996) have concluded otherwise, we felt it necessary to re-visit the issue of oocyte fragmentation and hopefully resolve this discrepancy by three approaches: (i) a careful analysis of DNA cleavage using the TUNEL assay for detection of DNA double-strand breaks in single oocytes; (ii) a single-cell assessment of caspase activation in fragmented oocytes; and (iii) a review of the currently available data regarding oocyte fragmentation with particular reference to the limitations and drawbacks of the various methods employed to assess apoptosis.
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Materials and methods |
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Collection and culture of oocytes
For studies of anti cancer drug-induced oocyte fragmentation, ovulation was stimulated in virgin female B6C3F1/CrlBR mice (Charles-River Laboratories, Wilmington, MA, USA) at 7–8 weeks of age with 10 IU of equine chorionic gonadotrophin (eCG; Professional Compounding Centers of America, Houston, TX, USA) followed by 10 IU of human chorionic gonadotrophin (HCG; Serono Laboratories, Norwell, MA, USA) 48 h later. Cumulus–oocyte-complexes were collected from the oviducts 16 h after HCG injection, and oocytes were denuded of cumulus cells by a 1 min incubation in 80 IU/ml hyaluronidase (Sigma Chemical Co, St Louis, MO, USA) followed by three washes with culture medium. Following isolation, oocytes were cultured in 0.1 ml drops of culture medium (8–10 oocytes per drop) under paraffin oil (Sigma Chemical Co), and incubated without (controls) or with 200 nmol/l doxorubicin (Sigma Chemical Co) for 24 h at 37°C in a humidified atmosphere of 5% CO2-95% air (Perez et al., 1997
). The culture medium used for all experiments was human tubal fluid (HTF; Irvine Scientific, Santa Ana, CA, USA) supplemented with 0.5% bovine serum albumin (BSA, Fraction V; Gibco-BRL Life Technologies, Grand Island, NY, USA). For studies of spontaneous oocyte fragmentation, oocytes were collected from FVB female mice (Taconic Laboratories, Germantown, NY, USA) at 7–8 weeks of age by ovulation stimulation as described above for B6C3F1 mice. This strain of mouse was selected since, unlike oocytes collected from B6C3F1 female mice, oocytes harvested from FVB female mice display a high rate of spontaneous fragmentation in vitro (see Results section). Oocytes were denuded of cumulus cells and cultured without doxorubicin as described for the B6C3F1 oocyte cultures. All studies involving animals described herein were approved by, and performed in strict accordance with, the guidelines of the Massachusetts General Hospital Institutional Animal Care and Use Committee and the NIH Guide for the Care and Use of Laboratory Animals.
Detection of apoptosis in oocytes by morphology
At the end of the incubation period without (spontaneous) or with doxorubicin (drug-induced), oocytes were fixed for 30 min in neutral-buffered 1% (w/v) paraformaldehyde prepared in 1x-concentrated Dulbecco's phosphate-buffered saline (PBS) and then checked by light microscopy for morphological changes characteristic of apoptosis (Kerr et al., 1972, 1994; see Results).
TUNEL analysis of DNA cleavage in oocytes
At the end of the incubation period without or with doxorubicin, oocytes were transferred into Tyrode's solution (Sigma Chemical Co) for 30 s at 37°C to remove the zona pellucida, washed quickly in PBS and then immediately fixed for 30 min in neutral-buffered 1% paraformaldehyde prepared in PBS containing 0.1 mg/ml polyvinyl alcohol (PVA, average molecular weight 30 000–70 000; Sigma Chemical Co). After fixation, oocytes were washed once more with PBS, transferred to Superfrost-Plus slides (Fisher Scientific, Pittsburgh, PA, USA) in small drops (10 oocytes/10 µl drop), and air-dried. Slides were heated at 65°C for 4 h, and then stored at 4°C until processed for in-situ DNA 3'-end-labelling as detailed previously from our laboratory (Tilly, 1994), with slight modifications. Briefly, slide-mounted oocytes were heated at 65°C for 30 min, immediately rehydrated through a graded ethanol series (absolute, 90%, 80% and 70% ethanol, 20 s each) to sterile water, and then treated with proteinase K (10 µg/ml; Sigma Chemical Co) at 37°C for 30 min followed by two washes in sterile water. To block for non-specific binding, slide-mounted oocytes were pre-incubated with 3% (w/v) BSA for 30 min at 20°C, and then pre-equilibrated with 1x-concentrated terminal deoxynucleotidyl transferase (TdT) reaction buffer (Boehringer-Mannheim, Indianapolis, IN, USA) for 20 min at 20°C. The TdT-mediated labelling reaction of DNA 3'-ends was performed by incubating the slide-mounted oocytes in the presence of 1.25 IU/µl TdT enzyme (Boehringer-Mannheim; with TdT reaction buffer and CoCl2 supplied with the enzyme) and 50 pmol/l fluorescein-labelled dUTP (Boehringer-Mannheim) at 37°C for 15 min in the dark. After 3'-end labelling, the slides were placed in 1x concentrated TE buffer (10 mmol/l Tris–HCl, 100 mmol/l EDTA, pH 8) to stop the reaction, and then rinsed several times with sterile water. Excess water was blotted away, mounting medium (Cytoseal 60; Stephens Scientific, Riverdale, NJ, USA) was added, and the slides were sealed with coverslips. The occurrence of DNA cleavage was assessed by fluorescence microscopy using a fluorescein filter.
Analysis of caspase activity in single oocytes
Stocks of the rhodamine-conjugated DEVD (Asp-Glu-Val-Asp) caspase substrate (PhiPhiLux) were prepared according to the manufacturer's instructions (OncoImmunin Inc, College Park, MD, USA), aliquoted and stored at –20°C until use. Oocyte cultures were conducted as described above with the exception that the caspase substrate was added to the microdrop cultures (final concentration of 180 nmol/l) at 23 h following the initiation of culture. The culture was then continued for 1 h more at 37°C, after which the oocytes were washed three times with cold (4°C) culture medium and then fixed in 1% paraformaldehyde containing 0.1 mg/ml PVA for 30 min at room temperature in the dark. The oocytes were washed once with PBS, transferred to Superfrost-Plus slides, sealed with mounting medium under coverslips and visualized by fluorescence microscopy using a rhodamine filter. As a negative control for the assay, B6C3F1 oocytes were pretreated for 30 min with 100 µmol/l of an irreversible and specific peptide inhibitor of caspases, zVAD-fmk (reviewed in Thornberry and Lazebnik, 1998; see also Perez et al., 1997), prior to addition of doxorubicin. Cultures were then continued for 23.5 h and oocytes processed for rhodamine fluorescence (DEVD-rhodamine cleavage) as described above.
In some experiments, oocytes were simultaneously analysed for chromatin localization using the DNA-binding dye, Hoechst 33342 (Sigma Chemical Co). Oocytes were prepared, cultured and fixed as described above for the caspase activity assay. After fixation, oocytes were washed once with PBS and transferred to Superfrost-Plus slides in a small volume of PBS, and then mixed with Hoechst 33342 (30 µl of a 1 mg/ml stock solution prepared in sterile water combined with 750 µl of 2.3% sodium citrate and 250 µl of 95% ethanol) to a final concentration of 30 µg/ml. Hoechst staining was carried out in the dark for 3 min at 37°C, after which the solution was carefully removed and replaced with mounting medium. The slides were covered with coverslips and oocytes were analysed by fluorescence microscopy for caspase activity (rhodamine filter) and chromatin localization (UV filter).
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Results |
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In contrast to the relatively high rates of spontaneous fragmentation of FVB oocytes incubated for 24 h (70%, n = 100), oocytes harvested from B6C3F1 female mice and incubated for 24 h displayed very low levels (3%, n = 100) of spontaneous fragmentation (Figure 1A
). For B6C3F1 oocytes, inclusion of 200 nmol/l doxorubicin in the culture medium caused 62% (n = 100) of the oocytes to undergo fragmentation (Figure 1B) (quantitative data not shown; see Perez et al., 1997; Bergeron et al., 1998). By morphology, oocyte fragmentation proceeded as an initial condensation of the oocyte cytoplasm (retraction of the oolemma from the zona pellucida) and the formation of membrane protuberances, followed by budding and ultimate fragmentation of the dead oocyte into membrane-enclosed vesicles of unequal sizes (i.e. apoptotic bodies) (Figure 1B and Figure 2; see also Tilly et al., 1997). These observations were consistent with all previous reports of murine oocyte fragmentation in vitro (see Van Blerkom and Davis, 1998; and Introduction).
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To confirm the occurrence of DNA cleavage in oocytes during fragmentation in vitro, the next set of experiments utilized the TUNEL assay to non-isotopically label free 3'-ends of the oocyte DNA with fluorescein-conjugated dUTP molecules. Fluorescence microscopy revealed no incorporation of the labelling nucleotide in the spindle of any intact oocyte from B6C3F1 (n = 50) or FVB (n = 30) female mice (Figure 1C
), whereas 33% (n = 30) of intact FVB oocytes showed a positive TUNEL reaction in the polar body only. In contrast, all fragmented oocytes (spontaneous for FVB, n = 90; drug-induced for B6C3F1, n = 50) displayed clear evidence of extensive DNA labelling as revealed by clumps of brightly-fluorescent chromatin in the oocyte cytoplasmic fragments (Figure 1D,E). The occurrence of DNA cleavage, as assessed by this approach, was consistent with previous results obtained using fluorescent dyes that intercalate into nucleic acids, allowing visualization of chromatin segregation during oocyte fragmentation in vitro (Perez et al., 1997).
To further confirm the role of apoptosis in fragmentation of ovulated oocytes in vitro, we analysed individual oocytes for caspase activity using a rhodamine-conjugated caspase substrate, DEVD, that once taken up by the cell fluoresces following specific cleavage by caspase-3-like enzymes. Intact B6C3F1 (n = 100) or FVB (n = 30) oocytes cultured for 24 h in the absence of the anti cancer drug (Figure 2A) exhibited extremely low levels of background fluorescence (necessitating the use of a digitized computer system to enhance the basal fluorescence intensity by 100-fold so that the oocytes could be viewed under the fluorescence microscope) (Figure 2B). By comparison, DEVD cleavage activity was clearly detectable, without computer-assisted enhancement of fluorescence intensity, in all fragmented oocytes evaluated (Figure 2E-H) regardless of the stimulus for fragmentation (spontaneous for FVB, n = 60; drug-induced for B6C3F1, n = 100). In those oocytes cultured for 24 h with doxorubicin that had not yet fragmented (Figure 2C), intense and punctate fluorescence associated with DEVD-cleavage activity was consistently noted (Figure 2D). As a confirmation of the fidelity of the caspase activity assay, B6C3F1 oocytes pretreated for 30 min with a specific and irreversible caspase inhibitor, zVAD-fmk (100 µM), prior to doxorubicin treatment for 24 h failed to exhibit rhodamine fluorescence (Figure 3D). Although oocytes co-treated with zVAD-fmk also failed to undergo fragmentation in response to doxorubicin, the oocytes appeared granular (Figure 3C) and were thus probably degenerating through a non-apoptotic pathway. Lastly, simultaneous analysis of individual doxorubicin-treated B6C3F1 oocytes for caspase activity (DEVD–rhodamine cleavage) and chromatin (Hoechst 33342 staining) revealed that the punctate fluorescence associated with caspase activity (Figure 3F) consistently co-localized near the spindle apparatus (Figure 3G) at the start of oocyte fragmentation (Figure 3E).
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Discussion |
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The recent report regarding prematurity of the conclusion reached by ourselves (Perez and Tilly, 1997
; Perez et al., 1997; Bergeron et al., 1998) and others (Takase et al., 1995; Fujino et al., 1996; Xu et al., 1997) that apoptosis is the mechanism responsible for in vitro fragmentation of ovulated oocytes was based on the inability of the investigators to correlate DNA cleavage by TUNEL or PS exposure by annexin-V staining with fragmentation of murine or human oocytes (Van Blerkom and Davis, 1998). This final conclusion (Van Blerkom and Davis 1998), is perplexing for several reasons, the first of which is that these authors state that fragmented mouse oocytes `exhibited features that grossly resembled apoptotic body formation'. When one considers the original descriptions of apoptosis (Kerr et al., 1972), this single feature should serve as irrefutable evidence for the occurrence of this form of cell death in ovulated oocytes since apoptosis is first and foremost defined as a well-conserved series of morphological changes resulting in cellular fragmentation (reviewed in Kerr et al., 1994; Hockenberry, 1995; Majno and Joris, 1995).
This notwithstanding, it is also unclear why Van Blerkom and Davis (Van Blerkom and Davis 1998) were unable to detect DNA cleavage by TUNEL analysis of fragmented oocytes since this particular endpoint has been reported by a number of other investigators in comparable analyses of murine oocyte degeneration (Takase et al., 1995; Fujino et al., 1996; Xu et al., 1997; Jablonka-Shariff and Olsen, 1998), including ourselves in the present study. One possible explanation may reside in the fact that procedural modifications of the TUNEL assay from that originally described (Gavrieli et al., 1992; Tilly, 1994), such as the fixation procedures, use of fluorescein-conjugated deoxynucleotides and removal of the zona pellucida, were required for the detection of apoptosis in single fragmented oocytes. Thus, the inconsistent results obtained by Van Blerkom and Davis (Van Blerkom and Davis 1998) regarding the lack of agreement between DNA cleavage and oocyte fragmentation may be more due to technical difficulties in the assay itself.
The second endpoint used (Van Blerkom and Davis 1998) as a `molecular marker' for apoptosis was annexin V-staining to detect `flipping' of PS from the inner to outer surface of the plasma membrane, a relatively new endpoint in the study of apoptosis (reviewed in van Engeland et al., 1998). In their hands, the absence of a correlation between annexin V-staining and oocyte fragmentation was again taken as evidence for the absence of apoptosis. Although it may be that Van Blerkom and Davis (Van Blerkom and Davis 1998) failed to detect a low level of PS extrusion in fragmented oocytes, another more likely explanation is that oocytes, upon initiating and progressing through the process of apoptosis, simply do not flip PS to the outer surface of the oolemma. Exposure of PS, although a marker of cell death in many systems, does not occur in all paradigms of apoptosis (Frey, 1997). The reason(s) why some cells, but not others, flip PS during apoptosis remains to be elucidated. However, this may be due to the absence or presence of aminophospholipid translocase or differences in calcium availability in different cell lineages, both of which are known to modulate the PS flipping mechanism (Bratton et al., 1997),
Such variability in whether or not a given molecular marker of apoptosis is truly related to the occurrence of apoptosis in all cells has been reported for other classically-accepted biochemical endpoints of apoptotic cell death. For example, internucleosomal DNA cleavage, long considered a hallmark of apoptosis by many investigators (Wyllie, 1980; reviewed in Schwartzman and Cidlowski, 1993; Walker and Sikorska, 1994), was found to be dispensable for apoptosis to proceed in some cell types (Cohen et al., 1992; Oberhammer et al., 1993) or in the same cell type under different experimental conditions (Flaws et al., 1995). In addition, proteolytic cleavage of the nuclear protein PARP, although widely used as a molecular marker of apoptosis in many cell types (reviewed in Rosen and Casciola-Rosen, 1997; Duriez and Shah, 1997), does not occur during apoptosis in all cell types (Inayat-Hussain et al., 1997; Boone and Tsang, 1998).
Importantly, however, activation of the enzyme(s) responsible for cleavage of PARP and other key intracellular proteins during apoptosis, namely caspase-3 (and closely related caspases), is an event conserved in essentially all apoptotic cell death paradigms (for review, see Alnemri, 1997; Rosen and Casciola-Rosen, 1997; Cryns and Yuan, 1998), including examples of invertebrate programmed cell death (Xue et al., 1996; Song et al., 1997; reviewed in Hengartner, 1996). In agreement with this, and the fact that apoptosis of ovulated murine oocytes is known to require the functional expression of at least caspase-2 (Bergeron et al., 1998), we consistently detected an induction of caspase activity in fragmented oocytes. Moreover, and in contrast to intact oocytes not exposed to the anti-cancer drug, doxorubicin-treated intact oocytes also exhibited DEVD-cleavage activity, suggesting that caspases are activated in oocytes prior to cellular budding and fragmentation. This is in agreement with previous reports that caspases are responsible for, and hence their activation precedes, many of the morphologic and biochemical changes that occur in cells undergoing apoptosis (Enari et al., 1998; Janicke et al., 1998; reviewed in Alnemri, 1997; Cryns and Yuan, 1998).
At the subcellular level, the punctate fluorescence associated with caspase activity that was observed to co-localize with chromatin in anti-cancer drug-treated intact oocytes is intriguing. Although the reason(s) for this remains to be elucidated, it is possible that the induction of caspase activity first occurs in association with mitochondria since these important organelles are known to be redistributed to, and thus accumulate around, the newly formed spindle apparatus upon maturation of oocytes to the metaphase II stage (reviewed in Küpker et al., 1998). Recent investigations have shown in somatic cells that the caspase-3 precursor molecule exists in both the cytosol and in the intermediate space in mitochondria (Mancini et al., 1998). Upon delivery of an apoptotic stimulus to the cells, there occurs a rapid loss of the mitochondrial pro-caspase-3 enzyme with a concomitant increase in caspase-3 activity (Mancini et al., 1998). These findings, coupled with a wealth of new data indicating that the final cellular decision for apoptosis initiation is made at the level of the mitochondrion (reviewed in Golstein, 1997; Marzo et al., 1998) with the ensuing activation of death effector caspases (Li et al., 1997), supports the hypothesis that the punctate caspase activity observed in intact oocytes prior to fragmentation may represent the initiation of the caspase cascade at the level of mitochondria surrounding the oocyte spindle apparatus. Alternatively, we cannot rule out the possibility that rhodamine, once freed from DEVD by active caspases, non-specifically accumulates around nuclear material. However, even if this is the case, the rhodamine fluorescence consistently noted in oocytes prior to and during their demise still indicates that caspases have been activated to cleave rhodamine from the DEVD caspase substrate peptide.
In summary, the points brought up and clarified in this article, combined with the new data presented regarding caspase induction and the fact that murine oocyte fragmentation in vitro is dependent upon the functional expression of several genes comprising the evolutionarily-conserved apoptotic cell death programme (drug-induced: Perez et al., 1997; Bergeron et al., 1998; spontaneous: Morita et al., 1999b), offer substantial evidence that fragmentation and death of ovulated murine oocytes in vitro is an unequivocal example of apoptosis.
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Acknowledgments |
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We would like to thank Dr R.A.Fissore (University of Massachusetts, Amherst, MA, USA) for initial discussions regarding the use of the DEVD-rhodamine substrate for caspase activity assessments in single oocytes, and Dr J.Yuan (Harvard Medical School, Boston, MA, USA) and Mr Sam Riley (Massachusetts General Hospital, Boston, MA, USA) for technical assistance with the photomicroscopy. This study was supported by Public Health Service grants from the U.S. National Institutes of Health to J.L.T. (R01-ES08430, R01-AG12279, R01-HD34226), by a grant from the Massachusetts General Hospital Fund for Medical Discovery to G.I.P. and by Vincent Memorial Research Funds.
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1 To whom correspondence should be addressed
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Submitted on August 21, 1998; accepted on February 10, 1999.
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