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Injection of Sperm Cytosolic Factor Into Mouse Metaphase II Oocytes Induces Different Developmental Fates According to the Frequency of [Ca²⁺]_i Oscillations and Oocyte Age¹

^a Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts 01003 ^b Instituto de Ciencias Biomedicas de Abel Salazar, Universidade Do Porto, Porto, Portugal

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Intracellular calcium ([Ca²⁺]_i) rises are a hallmark of mammalian fertilization and are associated with normal activation of embryonic development. Injection of mammalian sperm cytosolic factor (SCF) into oocytes has been shown to trigger [Ca²⁺]_i rises similar to those observed during fertilization, and to initiate normal embryonic development. However, Ca²⁺ release has also been shown to be associated with cell death, but the mechanisms of the detrimental effects of Ca²⁺ stimulation on development have not yet been investigated. Thus, studies were undertaken using SCF to test the effects of [Ca²⁺]_i oscillations on oocyte activation in freshly ovulated and aged oocytes. Injections of 1 mg/ml SCF into freshly ovulated mouse metaphase II oocytes, which evoked Ca²⁺ responses with low frequency and short duration, induced normal activation and cleavage to the two-cell stage. Conversely, injection of 15 mg/ml SCF, which triggered high-frequency and persistent Ca²⁺ responses, induced abnormal activation that was characterized by abnormal chromatin configurations, inhibition of DNA synthesis, and lack of first mitotic spindle assembly. More importantly, fertilization-like Ca²⁺ responses induced by injection of 1 mg/ml SCF triggered cell death, rather than activation, in in vitro-aged oocytes. These oocytes exhibited extensive cytoplasmic and DNA fragmentation that was accompanied by activation of protein caspases, all of which are signs of apoptotic cell death. Fewer similarly aged oocytes that were either unstimulated or activated with 7% ethanol underwent fragmentation. Together, these results suggest that [Ca²⁺]_i oscillations are required to activate freshly ovulated oocytes, but if initiated at abnormally high frequency and duration or if induced in aged oocytes, the [Ca²⁺]_i oscillations may trigger premature termination of embryonic development.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Mammalian oocytes are ovulated at the metaphase stage of the second meiotic division (MII), and they remain arrested in this stage until fertilization. Fertilization induces oocyte activation and the initiation of embryonic development. Events characteristic of oocyte activation include cortical granule exocytosis, resumption of meiosis and extrusion of the second polar body, pronuclear formation, and the first mitotic cleavage. To induce activation, the sperm relies on the generation of intracellular calcium ([Ca²⁺]_i) oscillations ([1, 2]; see [3] for review). Although these oscillations are detectable in oocytes of all mammalian species studied to date, their frequency appears to be species-specific. Fertilized mouse and hamster oocytes show [Ca²⁺]_i rises every 2–10 min [4] while bovine, porcine, and human oocytes have longer intervals between rises ([5–7]; see [8] for review). The signaling mechanism(s) by which the sperm induces [Ca²⁺]_i oscillations in mammalian oocytes is not known. However, it has been shown that injection of sperm cytosolic factor (SCF), obtained from sperm of several species, is able to induce Ca²⁺ responses similar to those observed during normal fertilization (see [9] for recent review). Injections of this factor are able to induce normal activation, initiate in vitro embryonic development to the blastocyst stage [10–12], and, when combined with injection of immature spermatogenic cells, produce live born pups [13]. Thus, SCF is an appropriate tool to further investigate the function and molecular targets of [Ca²⁺]_i oscillations in the initiation of development.

In mammalian oocytes, the role of [Ca²⁺]_i oscillations has been investigated exclusively in the context of initiation of development. Ca²⁺ responses, however, have been associated with cellular necrosis [14] and, more recently, with the initiation of programmed cell death/apoptosis in numerous cell types and in response to a variety of stimuli [15, 16]. Programmed cell death/apoptosis is morphologically characterized by nuclear condensation with subsequent chromatin degradation, cellular deformation and shrinkage, and the appearance of cell protuberances on the cell surfaces that are released as fragments [17]. Programmed cell death/apoptosis has also been reported to occur in the female gonad and gametes (for recent review see [18]). In the ovary, for example, the number of oogonia and oocytes during fetal and postnatal development appear to be regulated by this mechanism [19–22], and ovulated oocytes aged in vitro or recovered from older females also appear to spontaneously die by apoptosis [23, 24]. Similarly, oocytes and embryos exposed to different stimuli or suboptimal culture conditions undergo the same fate [25–29].

The genes and proteins involved in the regulation and execution of the apoptotic program, including the caspase family of proteases (for review see [30, 31]) and the anti- and pro-apoptotic members of the Bcl-2 family of proteins [32, 33], are expressed in mammalian oocytes and embryos [28, 34, 35]. However, the signaling mechanisms that trigger apoptosis in oocytes and embryos have not been extensively investigated and remain to be elucidated. In the present work, we determined whether [Ca²⁺]_i oscillations, which are the normal activating signal during fertilization, could induce abnormal activation and apoptosis in oocytes. To carry out these studies, [Ca²⁺]_i oscillations were induced in freshly ovulated oocytes at higher than normal frequency by injection of high concentrations of SCF. In addition, fertilization-like oscillations were induced in aged oocytes, which are less developmentally competent. Both freshly ovulated and aged oocytes were assayed for evidence of apoptosis.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Oocyte and Egg Recovery

Germinal vesicle (GV)-stage oocytes were obtained by mincing the ovaries of 6- to 12-wk-old unstimulated female CD-1 mice. GVs that were surrounded by several layers of granulosa cells were selected for further use and were stripped by using a small bore pipette before microinjections. MII oocytes were collected from the oviducts of female mice stimulated with 5 IU of eCG (Sigma, St. Louis, MO) and induced to ovulate 48 h later by injection of 5 IU of hCG (Sigma). Fertilized embryos were obtained by mating the females immediately after the hCG injection. Embryos and MII oocytes were collected 14–15 h post-hCG into a Hepes-buffered solution (Tyrode's lactate [TL]-Hepes) supplemented with 10% heat-treated fetal calf serum (FCS; Gibco, Grand Island, NY). Cumulus cells were removed by a brief incubation in bovine testis hyaluronidase (Sigma). MII oocytes and embryos were cultured in 50 µl of potassium simplex optimized medium (KSOM; Specialty Media, Lavallette, NJ) under paraffin oil at 36.5°C in 5% CO₂ until the time of injection. Morphological evaluation of oocytes and embryos was performed under dissecting microscopes. Only intact MII oocytes that had extruded the first polar body were used in the course of these studies.

Microinjection Technique and Parthenogenetic Activation

Microinjection procedures were performed according to the method of Wu et al. [36]. Oocytes or embryos were placed in a 50-µl drop of TL-Hepes supplemented with 2.5% sucrose (w:v) near a 2-µl drop of SCF (1.0 mg/ml protein concentration and/or 15 mg/ml) or fura-2 dextran (fura-2 D, dextran 10 kDa; Molecular Probes, Eugene, OR) under paraffin oil. The injections were performed using a Nikon Diaphot microscope (Nikon, Inc., Garden City, NY) and Narishige manipulators (Medical Systems Corp., Great Neck, NY). A picoliter injector (PLI-100; Medical Systems Corp.) was used to inject all reagents into the cytoplasm of oocytes and zygotes by pneumatic pressure. The amount of injected solution was 5–10 pl [37], resulting in a final intracellular concentration of approximately 1–3% of the concentration in the injection pipette.

Injections of SCF were intended to induce activation of MII oocytes and were completed before 17 h post-hCG. Pronuclear formation was observed 5 h later (22 h post-hCG), and cleavage after overnight culture. The low concentrations of SCF (1 mg/ml) were chosen because, in preliminary studies, it triggered fertilization-like oscillations, induced normal activation events, and promoted development to the blastocyst stage. Each injection of SCF represented approximately 2.5–5 sperm equivalents [38]. The high concentration of SCF (15 mg/ml) was chosen because, in preliminary studies, it induced Ca²⁺ responses with abnormally high frequency, and the effects of this pattern of [Ca²⁺]_i rises on oocyte activation events and embryo development was not known. Injections of 15 mg/ml SCF into GV-stage and fertilized zygotes were intended to evaluate toxic effects of SCF, and these cells were observed approximately 14–16 h later for progression to MII or cleavage to the 2-cell stage, respectively. In addition, we have determined that injection of 15 mg/ml BSA, serum proteins, or brain extracts into oocytes failed to induce Ca²⁺ release or to induce any changes in the oocytes, indicating that injection of high protein concentrations alone does not affect oocyte viability ([36] and data not shown).

Ethanol was used to activate eggs that were aged in vitro for 24 h (39–40 h post-hCG). MII aged oocytes were exposed for 7 min to a 7% ethanol solution in TL-Hepes + BSA (3 mg/ml), washed three times in TL-Hepes + BSA, and cultured in KSOM.

Fluorescence Recordings and Ca²⁺ Determination

Oocytes and zygotes received injections of the fluorescent dye fura-2 dextran, and Ca²⁺ values were monitored using a Nikon Diaphot microscope fitted for fluorescence measurements as described previously [36]. [Ca²⁺]_i concentrations, R_min, and R_max were calculated according to the methods of Grynkiewickz et al. [39] and Poenie [40], as described before [36]. Oocytes and zygotes were individually monitored for [Ca²⁺]_i levels in a 50-µl drop of medium placed on a glass coverslip sealed over an opening in the bottom of a culture dish and covered with paraffin oil. After fluorescence baseline values were established, oocytes and embryos were given injections of SCF, and fluorescence ratios were obtained every 4 sec, after 1-sec readings at each wavelength, for 30 min. In some experiments, the recordings were stopped for 2 h and reinitiated for 30 min after the interruption.

Immunofluorescence and DNA Staining

DNA replication in zygotes/MII oocytes was evaluated by assessing incorporation of clorodioxyuridine (ClDU; Sigma). Zygotes/MII oocytes were incubated for 3 h in 50-µl drops of KSOM containing 50 µM ClDU at 36.5°C in the dark approximately 9 h postinjection or postfertilization (estimated time). After culture in ClDU, embryos/MII oocytes were rinsed twice in Dulbecco's PBS containing 3 mg/ml polyvinylpyrrolidone (Sigma), permeabilized in cold methanol for 10 min, and fixed in 3.7% paraformaldehyde in PBS for 30 min at 36.5°C. After three washes (10 min each) in PBS, the embryos/MII oocytes were placed in 4 N HCl at 36.5°C for 30–45 min to denature the DNA [41], and then washed extensively in PBS containing 0.05% Tween (PBS-T). To inhibit nonspecific binding of antibody, zygotes/MII oocytes were incubated for 30 min in 10% normal goat serum. Immunodetection of ClDU was performed by indirect immunofluorescence using a monoclonal antibody (clone BU-1, #RPN202; Amersham, Arlington Heights, IL) for 1 h at room temperature.

SCF-injected MII oocytes to be evaluated for spindle formation by -tubulin labeling were permeabilized and fixed in the same manner and labeled for 2 h at 36.5°C with a 1:100 dilution of anti--tubulin monoclonal antibody (Sigma; #T9026) in 10% normal goat serum. After exposure to the primary antibody, zygotes/MII oocytes labeled either for DNA synthesis or for tubulin were extensively washed in PBS-T and incubated with a goat-anti mouse Cy3-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA) for 30 min at room temperature. After several washes, zygotes/MII oocytes were mounted in 90% glycerol in PBS supplemented with 1.9 mM Hoechst 33258 (Sigma) to visualize chromatin.

To assess DNA morphology after injection of SCF, oocytes were either incubated in Hoechst 33342 (Sigma; 1 µg/ml in TL-Hepes) for 15 min or fixed with 3.7% paraformaldehyde in PBS supplemented with 0.1% Triton-X, washed three times in PBS, and then mounted as described above. Slides were examined using a Zeiss epifluorescence microscope (Carl Zeiss, Inc., Thornwood, NJ) and photographed with 1600 ASA film.

Assay of Caspase Activity

The PhiPhilux kit (OncoImmunin Inc., College Park, MD), a rhodamine-conjugated DEVD (Asp-Glu-Val-Asp) caspase substrate, was used to detect the activity of group II caspases in fragmented and nonfragmented MII oocytes and in zygotes, essentially as described by others [28, 29]. Freshly ovulated (15 h post-hCG) and aged (40 h post-hCG) MII oocytes either uninjected or injected with 1 mg/ml SCF were incubated in 50-µl drops of KSOM containing the synthetic caspase substrate at a final concentration of 5 µM for 4 h at 37°C under 5% CO₂. After incubation, oocytes/zygotes were washed at least three times in TL-Hepes supplemented with FCS, placed in a microdrop on a coverslip, and visualized by fluorescence microscopy. In the presence of caspase activity, the caspase substrate is cleaved, which results in a metabolite that fluoresces red.

Sperm Cytosolic Factor Preparation

SCF was prepared from boar semen as previously described [36, 38, 42]. Briefly, semen samples were washed twice with TL-Hepes medium, and the sperm pellet was resuspended in a solution containing 75 mM KCl, 20 mM Hepes, 1 mM EDTA, 10 mM glycerophosphate, 1 mM dithiothreitol, 200 µM PMSF, 10 µg/ml pepstatin, and 10 µg/ml leupeptin, pH 7.0. The resulting suspension was lysed by sonication for 30–35 min at 4°C (XL2020; Heat Systems Inc., Farmingdale, NY). The lysate was then centrifuged twice at 10 000 x g, and the supernatants were collected and ultracentrifuged at 100 000 x g for 1 h at 4°C. Ultrafiltration membranes (Centricon 30; Amicon, Beverly, MA) were used to wash and concentrate the extracts to 60 mg/ml of protein. The crude sperm extracts were then precipitated by exposure to a saturated solution of ammonium sulfate (50% final ammonium sulfate concentration) and centrifuged at 10 000 x g for 15 min at 4°C, and the precipitates were collected and stored at -80°C until use. Protein concentrations were determined using a Sigma kit for protein determinations.

Statistics

Statistical comparisons between treatment and control groups were carried out using the chi-square test. The data in Table 2 were compared by using Student's t-test. All comparisons were performed using the JMP IN software (SAS Institute, Cary, NC). In all cases, significance was at P < 0.05. Experiments were repeated at least three different times, and the number of oocytes used per experiment is indicated in the Results section and in the tables.

	RESULTS

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High Concentrations of SCF Induced Abnormal Activation

Our preliminary studies have shown that injection of 15 mg/ml SCF did not support development to the blastocyst stage (data not shown). To determine whether this detrimental effect on development was due to abnormal activation events, mouse MII oocytes were given injections of 1 mg/ml and 15 mg/ml SCF, and they were observed for evaluation of pronuclear formation, chromatin configuration, and cleavage to the two-cell stage. Fertilized 2-cell-stage embryos exhibited normal nuclei and chromatin configurations (Fig. 1, A and B). Similarly, injection of 1 mg/ml SCF induced normal rates of pronuclear formation and cleavage (Table 1), and the nuclei of these blastomeres exhibited normal decondensed DNA (Fig. 1, C and D). Conversely, oocytes injected with 15 mg/ml SCF showed significantly lower rates of pronuclear formation and cleavage (Table 1, P < 0.05). In addition, the DNA appeared to be poorly decondensed, with chromatin accumulations preferentially around the nucleoli and in the periphery of the pronuclei (Fig. 1, E–H). These oocytes, however, did not show obvious signs of DNA or cytoplasmic fragmentation (Fig. 1, E–H), although they exhibited abnormal cytoplasmic shapes.

View larger version (104K): [in this window] [in a new window]   FIG. 1. Injection of 1 mg/ml SCF into MII oocytes induced activation and cleavage to the 2-cell stage, but injection of 15 mg/ml SCF induced abnormal activation. Phase-contrast (left panels) and DNA fluorescence microscopy (right panels) of in vivo-fertilized 2-cell-stage embryos (A, B), and 1 mg/ml (C, D) and 15 mg/ml SCF- (E–H) injected oocytes, after overnight culture. Oocytes injected with 1 mg/ml SCF (C, D) exhibited normal cytoplasmic and nuclear morphology, but oocytes injected with 15 mg/ml SCF exhibited abnormal cytoplasmic morphology (E, G) and condensed DNA around the nucleoli (F, H)

Injection of High Concentrations of SCF Inhibited DNA Synthesis and Disturbed Spindle Assembly

To determine whether the abnormal pronucleus configurations observed in oocytes given injections of 15 mg/ml SCF were functionally inactive, we examined their ability to replicate DNA. Oocytes were given injections of either 1 mg/ml or 15 mg/ml SCF, cultured for 9 h, and then incubated for 3 h in the presence of ClDU. Unactivated MII oocytes did not show ClDU incorporation (Fig. 2A), but fertilized embryos that were used as positive controls exhibited normal DNA synthesis (Fig. 2B; n = 23 of 23 oocytes). DNA replication could also be detected in all oocytes injected with 1 mg/ml SCF (Fig. 2C; n = 36 of 36 oocytes). The replication sites were brightly stained and diffusely distributed in the nucleoplasm (Fig. 2C). In contrast, the majority of oocytes injected with 15 mg/ml SCF did not show detectable DNA synthesis activity (Fig. 2D; n = 5 of 25 oocytes showed a faint signal).

View larger version (74K): [in this window] [in a new window]   FIG. 2. Injection of 15 mg/ml SCF inhibited DNA synthesis in MII oocytes. Unactivated MII oocytes showed no DNA synthesis (A), but DNA synthesis was observed in in vivo-fertilized zygotes (B) and in oocytes injected with 1 mg/ml SCF (C). Oocytes injected with 15 mg/ml SCF did not exhibit DNA synthesis (D)

Injection of solutions containing high Ca²⁺ concentrations has been shown to have a detrimental effect on spindle organization [43]. Thus, the effects of SCF on spindle assembly in oocytes were investigated 18–19 h postinjection of SCF. As shown in Figure 3, A and B, injection of 1 mg/ml SCF induced the formation of the first mitotic spindle (n = 22 of 28 oocytes). However, oocytes injected with 15 mg/ml SCF failed to form a spindle (Fig. 3, C and D; n = 0 of 18 oocytes).

View larger version (83K): [in this window] [in a new window]   FIG. 3. Injection of 15 mg/ml SCF into MII oocytes disturbed formation of the first mitotic spindle. Oocytes were injected with 1 mg/ml or 15 mg/ml SCF, fixed 18–19 h post-treatment, and double-stained with the DNA-specific Hoechst dye (B, D) and with Cy-3 labeled anti-tubulin antibody. The first mitotic spindle (A) and respective chromatin organized in a metaphase plate of an oocyte injected with 1 mg/ml SCF (B). Oocytes injected with 15 mg/ml failed to form a spindle (C), and the chromatin was disorganized and condensed (D)

Injection of High Protein Concentrations of SCF Induced Long-Lasting [Ca²⁺]_i Oscillations

To determine whether the detrimental effects on activation induced by injection of high concentrations of SCF are related to the elicited Ca²⁺ responses, MII oocytes were given injections of SCF as indicated above, and the Ca²⁺ responses were monitored. Injection of 1 mg/ml SCF induced [Ca²⁺]_i rises in all injected freshly ovulated oocytes, and their frequency appeared to decrease by 30 min postinjection (Table 2 and Fig. 4, A and B). Injection of 15 mg/ml SCF, on the other hand, induced very high-frequency [Ca²⁺]_i responses, which were unaltered for the first 30 min of recordings. Moreover, relatively high-frequency [Ca²⁺]_i oscillations were observed after an interval of 2 h in the recordings (Table 2 and Fig. 4, C and D).

View larger version (27K): [in this window] [in a new window]   FIG. 4. SCF-induced Ca2+ responses were dependent on protein concentration and cell-cycle stage. Injection of 1 mg/ml SCF induced fertilization-like [Ca2+]i oscillations in freshly ovulated oocytes (A), that were not detectable 2 h postinjection (B). Injection of 15 mg/ml SCF induced high-frequency [Ca2+]i oscillations that were still present 2 h postinjection (C, D, respectively). Injection of 15 mg/ml SCF induced short-lived Ca2+ responses in fertilized zygotes (E). Normal responses were observed in aged oocytes injected with 1 mg/ml SCF (F)

Injection of High Concentrations of SCF Into GV Oocytes or Zygotes Did Not Affect Oocyte Maturation or Cleavage to the Two-Cell Stage, Respectively

To examine whether the detrimental effects induced by injection of high protein concentrations of SCF into MII oocytes were due to a component(s) other than that responsible for triggering Ca²⁺ release, GV oocytes and fertilized zygotes were given injections of 15 mg/ml SCF. Injection of SCF did not have a significant effect on the progression of GV oocytes to the MII stage, as 41 of 57 (72%) injected and 29 of 38 (76%) uninjected GVs progressed to MII (P > 0.05). Similarly, 29 of 37 (78%) SCF-injected zygotes cleaved to the 2-cell stage as compared to 20 of 22 (91%) uninjected control embryos (P > 0.05). In GV oocytes, we have previously demonstrated that injection of SCF induces high-frequency oscillations [36], and, from the above results, it appears to have no detrimental effect on oocyte maturation. In pronuclear-stage embryos, injection of SCF initiated a single rise in [Ca²⁺]_i or low-frequency [Ca²⁺]_i oscillations that lasted for less than 15 min (Fig. 4E; 5 of 5 monitored zygotes).

Fertilization-Like Oscillations Triggered by SCF Induced Cytoplasmic and Nuclear Fragmentation in Aged Oocytes

Freshly ovulated mammalian oocytes can be induced to undergo programmed cell death/apoptosis by exposure to several chemical compounds [25, 44]. To determine whether a fertilization-like Ca²⁺ signal may induce apoptosis when imposed on aged oocytes, which are less developmentally competent [45], oocytes were collected 15 h post-hCG, aged in vitro for an additional 22–24 h, and injected with 1 mg/ml SCF. These oocytes were then observed for signs of activation within 7 h postinjection. Aged oocytes injected with 1 mg/ml SCF initiated normal Ca²⁺ responses (Fig. 4F). However, within 3 h of the injection, the majority of these oocytes showed signs of cytoplasmic fragmentation, and by 7 h, 65 of 71 oocytes (Table 3; Fig. 5C) exhibited severe fragmentation. Control uninjected aged oocytes, however, showed a very low rate of fragmentation (Fig. 5A; n = 5 of 60 oocytes), which is similar to data previously published for oocytes of this strain [34]. All freshly ovulated oocytes injected with the same concentration exhibited pronuclear formation (Fig. 5B; Table 3) and cleaved normally. We then tested whether the same rate of fragmentation was induced by triggering a single [Ca²⁺]_i rise. Aged oocytes treated with 7% ethanol for 7 min, a standard parthenogenetic activation procedure known to trigger a [Ca²⁺]_i rise [46, 47], showed significantly lower rates of fragmentation (Table 3). In SCF-injected aged oocytes, the cytoplasmic fragmentation was accompanied by DNA cleavage, and DNA fragments were observed in several of the cytoplasmic bodies (Fig. 5D). The few control untreated aged oocytes that spontaneously fragmented and aged oocytes exposed to ethanol that fragmented also showed DNA fragmentation (data not shown).

View larger version (105K): [in this window] [in a new window]   FIG. 5. Injection of 1 mg/ml SCF into aged MII oocytes induced cytoplasmic and DNA fragmentation. Uninjected MII aged oocytes (40 h post-hCG) showed no signs of fragmentation (A). Freshly ovulated oocytes exhibited pronuclear formation in response to 1 mg/ml SCF (B), but aged oocytes (40 h post-hCG) were fully fragmented 5 h postinjection (C). Hoechst staining of fragmented aged oocytes revealed DNA cleavage within 3 h postinjection (D)

We then determined whether the observed oocyte fragmentation was accompanied by activation of caspases, which are the known executors of the apoptotic program. SCF-injected and fragmenting oocytes showed widespread stimulation of caspase activity as detected with the PhiPhiLux kit (Fig. 6, E and F; n = 21 of 21 oocytes). In nonfragmenting aged oocytes (Fig. 6, A and B; n = 10 of 11 oocytes) and freshly ovulated oocytes injected with 1 mg/ml SCF (Fig. 6, C and D; n = 15 of 16 oocytes), caspase activity was exclusively associated with the polar body as previously reported by others [28, 29].

View larger version (82K): [in this window] [in a new window]   FIG. 6. Fragmented, SCF-injected MII aged oocytes exhibited widespread caspase activity. Phase contrast images of uninjected MII aged oocyte (40 h post-hCG) and freshly ovulated oocyte (16 h post-hCG) injected with 1 mg/ml SCF show no signs of cytoplasmic fragmentation (A, C), and caspase activity was solely present in the polar body (B, D). Aged oocytes (40 h post-hCG) injected with 1 mg/ml SCF showed cytoplasmic fragmentation (E) and exhibited clear evidence of extensive caspase activity (F)

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The results presented in this study show that 1) fertilization-like [Ca²⁺]_i oscillations induced by injection of 1 mg/ml SCF evoked normal activation in freshly ovulated mouse oocytes; 2) the initiation of abnormally high frequency and persistent [Ca²⁺]_i rises induced by injections of 15 mg/ml SCF triggered abnormal activation events that led to cell cycle arrest; 3) the induction of fertilization-like oscillations in in vitro-aged oocytes initiated severe cytoplasmic and DNA fragmentation with caspase activation characteristic of apoptosis; and 4) the exposure of aged oocytes to 7% ethanol induced a much less severe degree of fragmentation, in considerably fewer oocytes. These results suggest a novel role for [Ca²⁺]_i oscillations in MII mouse oocytes. In freshly ovulated oocytes, [Ca²⁺]_i oscillations are required to induce activation, but in aged and less developmentally competent oocytes, oscillations induce cessation of development by triggering apoptosis.

Mammalian MII oocytes exhibit species-specificity in regard to the frequency and duration of fertilization-associated [Ca²⁺]_i oscillations [8]. The significance of these patterns and the developmental consequences caused by their modification have not been thoroughly investigated. In mouse oocytes, it has been shown that increasing the frequency and duration of [Ca²⁺]_i rises induced by electrical stimulation resulted in higher rates of oocyte activation and decreased time to pronuclear formation [48, 49]. Similarly, additional Ca²⁺ stimulation imposed by a variety of parthenogenetic treatments resulted in higher developmental and implantation rates [50]. However, excessive [Ca²⁺]_i release has been shown to have detrimental effects on cell functions and survival [15, 51]. In the study reported here, we demonstrated that excessive Ca²⁺ release is detrimental for development: high-frequency [Ca²⁺]_i oscillations induced abnormal activation events including inhibition of DNA synthesis, lack of spindle assembly, and cell cycle arrest. Although the amounts of injected SCF in these experiments were not physiological, it is important to note that polyspermy has been shown to change the frequency of oscillations in mammalian oocytes [4, 52, 53]. Furthermore, in oocytes of the nemertean worm Cerebratulus lacteus, abnormally persistent oscillations, presumably caused by polyspermy, induced delayed or permanently inhibited cell cleavage [54]. In addition, it is important to point out that the success of intracytoplasmic sperm injection and cloning techniques has resulted in an increased use of Ca²⁺ agonists to induce oocyte activation. Our results show that eliciting persistent [Ca²⁺]_i oscillations with higher than normal frequency does not necessarily result in better activation and, in actuality, may have adverse effects on development. We cannot rule out, however, that some of the detrimental effects caused by injection of high protein concentration SCF may be due, at least in part, to deleterious acrosomal contents present in our SCF preparations.

Oocyte aging has also been shown to have detrimental effects on embryo development. Marston and Chang [45] demonstrated that oocytes could be fertilized many hours after ovulation without significant increases detected in the rates of polyspermy. Delaying insemination, however, resulted in a steady decrease in embryo development characterized by an increase in the rate of fragmentation/degeneration of embryos. Other investigators published similar results, and this outcome was attributed to postovulatory deterioration of oocytes [55, 56]. More recently, it was shown that mouse oocytes aged in vitro underwent spontaneous fragmentation and that these oocytes exhibited many characteristics of apoptosis such as DNA fragmentation as assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) analysis [23, 24]. This fragmentation was more pronounced in oocytes of older females, which also exhibited significantly lower levels of fertilization [24]. Consistent with these findings, human preimplantation embryos also appear to be disposed of by apoptosis [57].

It appears, then, that fertilization can initiate two developmental programs, normal development or apoptosis, according to the age of oocytes. We speculated that the same fertilizing Ca²⁺ signal was responsible for both outcomes and tested this possibility by inducing the same frequency of [Ca²⁺]_i oscillations in freshly ovulated and in vitro-aged oocytes. In freshly ovulated oocytes, [Ca²⁺]_i oscillations induced normal activation and cleavage, but in aged oocytes they induced oocyte fragmentation that was accompanied by caspase activation and DNA fragmentation. In vitro-aged oocytes exposed to ethanol also exhibited fragmentation, although the number of oocytes and rate of fragmentation were significantly lower than those observed in the presence of multiple [Ca²⁺]_i rises. These results are in agreement with previously published results that demonstrated that ethanol causes modest fragmentation of in vitro-aged oocytes [58]. Together, these data suggest that in mouse oocytes, [Ca²⁺]_i oscillations, in addition to being the normal activating signal, may also serve to ensure the demise of embryos that arise from aged and less developmentally competent oocytes.

Although the molecular pathway(s) that makes aged oocytes susceptible to apoptosis remains to be elucidated, changes in the expression level of gene products of members of the Bcl-2 family are probably important in determining the oocyte's fate. For example, it has been reported that mouse embryos undergoing fragmentation exhibit increased expression of pro-apoptotic (Bad and Bcl-xS) members of the Bcl-2 family of proteins and decreased presence of anti-apoptotic (Bcl-2) members [34]. Consistent with these findings, in vivo-aged mouse MII oocytes appear to have fewer Bcl-2 transcripts [28, 35], and bovine oocytes lacking developmental competence exhibited decreased expression of the Bcl-2 protein [59]. Furthermore, genetic manipulations of several components of the apoptotic machinery have shown profound effects in the number of oocytes at birth and susceptibility to chemically induced apoptosis. For instance, Bcl-2-deficient mice are fertile, but they have a significantly lower number of primordial follicles, and many of these lack oocytes [19]. Conversely, mice deficient in Bax (a pro-apoptotic Bcl-2 family member) [22] and caspase-2 (an initiator/effector protease) [60] exhibit an overabundance of primordial germ cells, and their oocytes were resistant to cell death when exposed to doxorubicin, a chemotherapeutic drug [29, 60]. Similarly, targeted overexpression of Bcl-2 in oocytes resulted in a greater number of healthy follicles in the ovary of transgenic females, and in oocytes more resistant to spontaneous and chemically induced apoptosis [61]. These data support the model originally proposed by Oltvai et al. [62] that the ratio of Bcl-2 to Bax (or anti-apoptotic to pro-apoptotic family members' ratio) is critical in determining whether cells die or survive. The same model has been suggested to operate in oocytes and embryos [28, 34, 63]. Thus, it is likely that as oocytes age, critical anti-apoptotic gene products are lost either by degradation or inactivation, and such changes change the fertilizing Ca²⁺ signal into an apoptosis-triggering signal.

The precise mechanism of action by which anti-apoptotic Bcl-2 family members prevent apoptosis is not known [33]. It has been reported, however, that Bcl-2 is expressed in, and may act on, several organelles including mitochondria, endoplasmic reticulum (ER), and the nucleus [64]. In the ER, which is the main intracellular Ca²⁺ store, Bcl-2 may be critical to maintain Ca²⁺ homeostasis, and this, in part, may contribute to its anti-apoptotic effects [65–67]. This function of Bcl-2 appears plausible since overexpression of this protein prevented cell death induced by ER Ca²⁺ depletion [66]. In addition, Bcl-2-overexpressing cells showed increased Ca²⁺ uptake into the ER, and this may be due, at least in part, to the observed up-regulation at the mRNA and protein levels of the sarcoplasmic/ER Ca²⁺ ATPase (SERCA) pump [68]. The Bcl-2 protein also appears to interact directly with the SERCA protein, and this may provide an additional method of modulation of intracellular [Ca²⁺]_i levels [68]. Thus, it is possible that aged oocytes, which may have lower levels of Bcl-2 or other anti-apoptotic family members, may also have decreased amounts of functional SERCA proteins. The latter may result in decreased intra-ER levels of Ca²⁺ or in oocytes less able to handle fertilization-like [Ca²⁺]_i rises, making aged oocytes more prone to apoptosis. It is interesting to note that aged oocytes exhibited fertilization-induced [Ca²⁺]_i rises with slower rates of increase and decrease [69]. Furthermore, increases in extracellular Ca²⁺ induced sustained [Ca²⁺]_i elevations in aged oocytes rather than increases in frequency as observed in young oocytes [69].

In summary, our data show that [Ca²⁺]_i oscillations are critical for the initiation of development. However, if induced at significantly higher frequency than normal, they may trigger abnormal activation and developmental arrest. In addition, we show that fertilization-like oscillations induced in aged oocytes trigger apoptosis. Elucidating the mechanism(s) that renders aged oocytes susceptible to apoptosis may lead to methods to significantly increase the development potential and viability of in vitro-generated embryos.

	ACKNOWLEDGMENTS

 
We thank Ms. Teru Jellerette for help in conducting some of the experiments and Drs. Sallie Smith and Robert T. Duby for critically reading the manuscript. We also want to thank the reviewers for many helpful suggestions.

	FOOTNOTES

 
First decision: 22 October 1999.

¹ These experiments were funded in part by a USDA NRI competitive grant (97–2919) to R.A.F and by funds from CSREES Hatch/USDA project #MAS734 to R.A.F.

² Correspondence. FAX: 413 545 6326; rfissore{at}vasci.umass.edu

Accepted: December 15, 1999.

Received: September 22, 1999.

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