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Regular Article |
a Department of Animal Sciences, University of Florida, Gainesville, Florida 32611-0910
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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16 cells on Day 5 after insemination to 41 or 42°C for 9 h increased the percentage of cells undergoing apoptosis. In addition, there was a duration-dependent increase in the proportion of blastomeres that were apoptotic when embryos were exposed to temperatures of 40 or 41°C, which are more characteristic of temperatures experienced by heat-stressed cows. Heat shock also increased caspase activity in Day 5 embryos. However, heat shock did not induce apoptosis in 2- or 4-cell embryos, nor did it increase caspase activity in 2-cell embryos. The apoptotic response of 8- to 16-cell-stage bovine embryos to heat shock depended upon the day after insemination that heat shock occurred. When 8- to 16-cell embryos were collected on Day 3 after insemination, heat shock of 41°C for 9 h did not induce apoptosis. In contrast, when 8- to 16-cell embryos were collected on Day 4 after insemination and exposed to heat shock, there was an increase in the percentage of cells undergoing apoptosis. Exposure of 8- to 16-cell embryos at Day 4 to a mild heat shock of 40°C for 80 min blocked the apoptotic response to a subsequent, more-severe heat shock of 41°C for 9 h. In conclusion, apoptosis is a developmentally acquired phenomenon that occurs in embryos exposed to elevated temperature, and it can be prevented by induced thermotolerance.
apoptosis, developmental biology, early development, embryo, stress
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In several species, deleterious effects of heat shock decrease as embryos advance in development. This has been shown in vivo in sheep [7], pigs [8], cattle [9], and rabbits [11] and in vitro for cattle [5, 12, 13]. Thus, the embryo acquires one or more thermoprotective responses as embryonic development proceeds.
One of the processes that may be involved in developmental acquisition of resistance to heat shock may be stress-induced apoptosis. Apoptosis plays a role in mammalian development as a quality control mechanism to eliminate cells that are damaged, nonfunctional, abnormal, or misplaced [14–16]. Cells severely damaged by stress that do not undergo apoptosis often become necrotic [17, 18].
TUNEL-positive embryos have been demonstrated in mouse [19–21], human [22, 23], and bovine [24–26] species. The occurrence of apoptosis in bovine embryos as determined by TUNEL staining has been shown to be developmentally regulated. Spontaneous apoptosis was first observed in bovine embryos at the 8- to 16-cell stage [24, 25]. This stage of development in cattle is coincident with the time of the major activation of the embryonic genome [1], and it is possible therefore that embryonic transcription may be involved in apoptotic responses in embryos.
Although apoptosis is known to occur in preimplantation embryos, there are few studies on extrinsic or intrinsic control systems for activation of apoptosis in preimplantation embryos or the ontogeny of such systems. Moreover, it is not known whether agents such as heat shock, which can induce apoptosis in many cell lines through activation of acid sphingomyelinase [27–29], also induce apoptosis in preimplantation embryos. The working hypothesis of this series of experiments was that heat-induced apoptosis is a developmentally regulated process. Experiments were performed to 1) determine whether heat shock can induce apoptosis in preimplantation embryos, 2) test whether heat-induced apoptosis in embryos depends upon stage of development, 3) evaluate whether heat shock-induced changes in caspase activity parallel patterns of apoptosis, and 4) ascertain whether exposure to a mild heat shock can protect embryos from heat-induced apoptosis. The last objective was conducted because of evidence that apoptosis in heat-shocked cells can be reduced by heat shock protein 70 (HSP70) [30, 31] or mild heat shock that presumably induces HSP70 synthesis [31–33].
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Essentially fatty-acid free (EFAF) BSA was purchased from Sigma Chemical Company (St. Louis, MO). Bovine steer serum was from Pel-Freez (Rogers, AR). Modified Tyrode solutions were obtained from Cell and Molecular Technologies (Lavallette, NJ) to prepare Hepes-Tyrode albumin lactate pyruvate (TALP), in vitro fertilization (IVF)-TALP and Sp-TALP [34]. Pituitary-derived FSH (Folltropin-V) was purchased from Vetrepharm Canada Inc. (London, ON, Canada). Percoll was from Amersham Pharmacia Biotech (Uppsala, Sweden). Frozen semen from various bulls was purchased from American Breeders Service (Madison, WI) or was donated by Select Sires Inc. (Rocky Mount, VA). Potassium simplex optimized medium (KSOM) was obtained from Cell and Molecular Technologies. The KSOM, which contains 1 mg/ml BSA, was modified on the day of use by adding an additional 2 mg/ml EFAF-BSA, 2.5 µg/ml gentamicin, essential amino acids (basal medium Eagle), and nonessential amino acids (minimum essential medium) purchased from Sigma. Oocyte collection medium was tissue culture medium 199 (TCM-199) with Hanks salts without phenol red and supplemented with 2% (v/v) bovine steer serum (containing 2 U/ml heparin), 100 U/ml penicillin-G, 0.1 mg/ml streptomycin, and 1 mM glutamine. Oocyte maturation medium was TCM-199 with Earle salts supplemented with 10% (v/v) steer serum, 22 µg/ml sodium pyruvate, 20 µg/ml FSH, 2 µg/ml estradiol-17ß, 50 µg/ml gentamicin, and an additional 1 mM glutamine.
The in situ cell death detection kit (fluorescein) and propidium iodide were obtained from Roche Diagnostics Corporation (Indianapolis, IN) and Sigma, respectively. Polyvinyl pyrrolidine (PVP) was purchased from Eastman Kodak Company (Rochester, NY). The Prolong Antifade Kit was obtained from Molecular Probes (Eugene, OR), RQ1 RNA-free DNase was from Promega (Madison, WI), and RNase A was from Qiagen (Valencia, CA). PhiPhiLux-G1D2 was obtained from OncoImmunin, Inc. (Gaithersburg, MD). Other reagents were purchased from Fisher (Pittsburgh, PA) or Sigma.
In Vitro Production of Embryos
Embryos were produced using procedures described earlier [35, 36] except that the culture medium for embryos was different. Briefly, cumulus-oocyte complexes (COCs) were obtained by slicing 2- to 10-mm follicles on the surface of the ovaries obtained from slaughtered cows (a mixture of beef and dairy cattle). COCs that had at least one layer of compact cumulus cells were washed two times and used for subsequent steps. Groups of 10 COCs were placed in 50-µl drops of oocyte maturation medium overlaid with mineral oil. For most experiments, COCs were matured for 22 h at 38.5°C in an atmosphere of 5% (v/v) CO2 in humidified air. A longer time for oocyte maturation (24–26 h) was used for the experiment with caspase activity for technical reasons to ensure that the fluorescence microscope required was available when needed. COCs were removed from maturation drops and washed one time in Hepes-TALP. For in vitro fertilization, groups of 30 COCs were transferred to 4-well plates containing 600 µl IVF-TALP and 25 µl PHE (0.5 mM penicillamine, 0.25 mM hypotaurine, and 25 µM epinephrine in 0.9% [w/v] NaCl) per well and fertilized with 
1 x 106 Percoll-purified spermatozoa. After 8–10 h at 38.5°C and 5% (v/v) CO2 in humidified air, presumptive zygotes were removed from fertilization wells, denuded of cumulus cells by vortexing in 30 µl of Hepes-TALP for 5 min in a microcentrifuge tube, washed two to three times in Hepes-TALP, and placed in groups of 25–30 in 50-µl drops of modified KSOM overlaid with mineral oil at 38.5°C and 5% CO2 (v/v) in humidified air. Embryos were harvested from culture at different times according to the specific experimental design. Two-cell embryos were harvested at 28–30 h after insemination; 4-cell embryos at 37–39 h; 8- to 16-cell embryos on Day 3 or 4 after insemination, and embryos 16 cells on Day 5 after insemination.
Using this system of embryo production, the cleavage rate ranged from 77% to 100% across all the experiments. In several replicates, one plate of embryos was not subjected to treatment or any staining procedure, and embryos were allowed to develop to the blastocyst stage to verify adequacy of culture conditions. In these plates, the percentage of putative zygotes developing to blastocyst by Day 8 after insemination ranged from 33% to 59%.
TUNEL and Propidium Iodide Labeling
The TUNEL procedure was used to detect DNA fragmentation observed in late stages of apoptosis. The terminal deoxynucleotidyl transferase enzyme is a DNA polymerase that catalyzes the transfer of a fluorescein isothiocyanate-conjugated dUTP nucleotide to a free 3' hydroxyl group present in DNA strand breaks. Embryos were removed from culture medium (modified KSOM) and washed four times in 100-µl drop of PBS (pH 7.4) containing 1 mg/ml PBS-PVP by transferring the embryos from drop to drop. Zona pellucida-intact embryos were fixed in a 100-µl drop of 4% (w/v) paraformaldehyde in PBS pH 7.4 for 1 h at room temperature, washed twice in PBS-PVP, transferred to a poly-L-lysine coated slide, and allowed to dry for 24 h at room temperature. Embryos were then washed twice by dipping the slide in a Coplin jar containing PBS-PVP (2 min/wash) and permeabilized in 0.5% (v/v) Triton X-100 containing 0.1% (w/v) sodium citrate for 1 h at room temperature. Positive controls were incubated with RQ1 RNase-free DNase (50 U/ml) at 37°C for 1 h. Embryos were washed in PBS-PVP and incubated with 50 µl of TUNEL reaction mixture (containing fluorescein isothiocyanate-conjugated dUTP), and the enzyme terminal deoxynucleotidyl transferase (as prepared by the manufacturer) for 1 h at 37°C in the dark. Negative controls were incubated in the absence of terminal deoxynucleotidyl transferase. Embryos were then incubated with RNase A (50 µg/ml) for 1 h at room temperature, followed by 0.5 µg/ml of propidium iodide for 30 min at room temperature. Embryos were washed four times in PBS-PVP to remove excess propidium iodide. A coverslip was mounted with 16-µl mounting medium containing Antifade as recommended by the manufacturer. Labeling was observed using a Zeiss Axioplan 2 fluorescence microscope with a dual filter. Each embryo was analyzed for total number of nuclei and number of TUNEL-labeled nuclei. Some embryos were also examined using a Bio-Rad 1024ES laser scanning confocal microscope. For fluorescein, an argon-ion laser adjusted to less than 560 nm was used, and for propidium iodide, a helium-neon laser adjusted to more than 560 nm was used. Images were obtained using a 30x objective, 10% power, and Z-steps of 0.5–1.0 µm.
Caspase Activity
PhiPhiLux-G1D2 is a fluoroprobe that incorporates the group II caspase-recognition sequence DEVD into a bifluorophore-derivated peptide that mimics the structural loop conformation present in native protease cleavage sites. Group II caspases include caspase 3, caspase 2, and caspase 7. In this molecule, the core peptide, GDEVDGI, is coupled to a molecule of rhodamine on each side of the cleavage site. The two rhodamines interact as a dimer and emit a stable blue-green fluorescence. Cleavage of the substrate disrupts this interaction between rhodamine moieties to result in enhanced green fluorescence (excitation peak 490 nm and emission peak 520).
To measure caspase activity, embryos were removed from culture medium and washed three times in 50-µl drops of prewarmed Hepes-TALP. Embryos were incubated in 25-µl microdrops of Hepes-TALP containing 5 µM PhiPhiLux-G1D2 at 39°C for 40 min in the dark. Negative controls were incubated in Hepes-TALP only. Following incubation, embryos were washed twice in 50-µl drops of Hepes-TALP and placed on poly-L-lysine coated slides and mounted with a coverslip. Caspase activity was determined immediately after the end of heat shock using a Zeiss Axioplan 2 fluorescence microscope with a 45x objective. Images were obtained using a Spot camera and software (Diagnostic Instruments, Inc., Sterling Heights, MI). Pictures were taken using a background subtract feature from the spot software, and digital images were stored as .tif files. Fluorescence intensity was analyzed using IPLab for MacIntosh version 3.5 (BioVision Technologies, Inc., Exton, PA). Each embryo represented a region of interest. Using the mouse, a circular draw function was manually performed for each region of interest, and the pixel intensity per unit area was determined.
Experiments
A series of six experiments was conducted to evaluate heat-induced apoptosis. For TUNEL analysis, embryos were collected at different times in development, transferred to a new drop of modified KSOM (15–30 embryos per drop; within a run, similar numbers of embryos per drop were used for each treatment), and maintained at 38.5°C for 24 h or exposed to various heat shock (40, 41, or 42°C) of 3- to 12-h duration. A heat shock of 42°C was used in the initial experiment to maximize probability of induction of apoptosis. In subsequent experiments, embryos were exposed to more physiological temperatures (40–41°C) that were similar to those experienced by heat-stressed cows. Heat-shocked embryos were then returned to 38.5°C. At 24 h after the initiation of heat shock, embryos were fixed in 4% (w/v) paraformaldehyde, transferred to a poly-L-lysine coated slide and saved at 4°C until analysis by TUNEL. For caspase activity, embryos were exposed to either 38.5°C or 41°C for 9 h. Immediately after the end of heat shock, caspase activity was determined as previously described. Details of specific experiments are described below.
Heat-induced apoptosis in bovine embryos 16-cell stage
Bovine embryos that were 16 cells were collected on Day 5 after insemination and transferred to a new drop of modified KSOM. Embryos were cultured at 38.5°C for 24 h or were heat shocked at 41 or 42°C for 9 h followed by 38.5°C for 15 h. Analysis by TUNEL was then performed. The experiment was replicated four times using 208 embryos (50–85 embryos/group).
Effect of magnitude and duration of heat shock on heat-induced apoptosis
The purpose of this experiment was to determine whether the degree of apoptosis was proportional to the severity of heat shock. This was accomplished by heat shocking embryos at two different temperatures for variable durations. Embryos 16 cells were collected on Day 5 after insemination and transferred to a new drop of modified KSOM (15–30 embryos per drop). Embryos were cultured at either 38.5°C for 24 h or were heat shocked at 40 or 41°C for 3, 6, or 9 h followed by 38.5°C for a total culture period of 24 h. Twenty-four hours after the initiation of heat shock, embryos were fixed in 4% paraformaldehyde, transferred to a poly-L-lysine coated slide, and saved at 4°C until analysis by TUNEL. The experiment was replicated four times using 342 embryos (30–70 embryos/group).
Heat-induced apoptosis in bovine embryos at the 2- or 4-cell stage Embryos at the 2- or 4-cell stage were collected at 28–30 and 37–39 h after insemination, respectively. Embryos were transferred to a new drop of modified KSOM and cultured at 38.5°C for 24 h or were heat shocked at 41°C for 9 h followed by 38.5°C for 15 h. Embryos were fixed in 4% paraformaldehyde, transferred to a poly-L-lysine coated slide and saved at 4°C until analysis by TUNEL. The experiment was replicated seven times using 491 embryos (94–146 embryos/group).
Heat-induced apoptosis in bovine embryos at the 8- to 16-cell stage on Day 3 after insemination Embryos at the 8- to 16-cell stage were collected on Day 3 after insemination and transferred to a new drop of modified KSOM. Embryos were then cultured at 38.5°C for 24 h or were heat shocked at 40 or 41°C for 9 h followed by 38.5°C for 15 h. At the end of culture, embryos were fixed and subjected to TUNEL analysis. The experiment was replicated three times using 188 embryos (52–64 embryos/group).
Induced thermotolerance in bovine embryos at the 8- to 16-cell stage on Day 4 after insemination Embryos at the 8- to 16-cell stage were collected on Day 4 after insemination and transferred to a new drop of KSOM (15–30 embryos per drop). Embryos were then subjected to four treatments: control (38.5°C for 24 h), mild heat shock (40°C for 80 min followed by 38.5°C), severe heat shock (41°C for 9 h followed by 38.5°C for 15 h), or thermotolerance (40°C for 80 min followed by 2 h at 38.5°C and 9 h at 41°C). At 24 h after initiation of temperature treatment, embryos were fixed in 4% paraformaldehyde, transferred to a poly-L-lysine coated slide and saved at 4°C until analysis by TUNEL. The experiment was replicated four times using 473 embryos (111–120 embryos/group).
Caspase activity in bovine embryos
Embryos at the 2-cell stage or 16-cell stage were collected 28–30 h after insemination or on Day 5 after insemination, respectively. Embryos were transferred to a new drop of modified KSOM and cultured at 38.5°C for 24 h or heat shocked at 41°C for 9 h followed by 38.5°C for 15 h. After heat shock, embryos were washed three times in a 50-µl drop of prewarmed Hepes-TALP and analyzed for caspase activity as described above. The experiment was replicated five times using 350 embryos. Caspase activity was quantified using 135 embryos produced in two replicates (22–42 embryos/group).
Statistical Analysis
Data were analyzed by least-squares analysis of variance using the General Linear Models procedure of SAS [37]. Percentage data were transformed using the arcsin transformation before analysis. Dependent variables were total cell number, percentage of apoptotic cells, and the arcsin of the percentage of cells displaying apoptosis. Independent variables varied according to the experimental design and included treatment, day (i.e., replicate), and stage of embryonic development. The mathematical model included main effects and all interactions. All main effects were considered fixed. Orthogonal contrasts and a means separation procedure of SAS called pdiff were performed when appropriate to determine differences between levels of individual treatments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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16 Cells
Figure 1, A–C, displays representative confocal digital images of embryos that were collected on Day 5 after insemination (if cell number was 16 cells), cultured at either 38.5°C for 24 h or at 41°C for 9 h and 38.5°C for 15 h, and then subjected to the TUNEL reaction. An embryo exposed to 38.5°C is shown in Figure 1A, whereas embryos exposed to 41°C are shown in Figure 1, B and C. An increase in the proportion of nuclei labeling positive for TUNEL (yellow in color) is apparent in the embryos exposed to 41°C. The percentage of cells undergoing apoptosis following heat shock was determined in several experiments. In the first experiment, exposure of embryos 16 cells to 41 or 42°C for 9 h increased (P < 0.05) the percentage of cells undergoing apoptosis 24 h after initiation of heat shock compared with embryos cultured at 38.5°C for 24 h (Fig. 2A). Heat shock also tended to reduce (P = 0.07) total number of cells per embryo 24 h after initiation of treatment (Fig. 2B). The effect of heat shock was similar for 41°C or 42°C.
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In a second experiment, embryos were exposed to temperatures more characteristic of rectal temperatures experienced by cows during heat stress. In addition, the effect of different durations of heat shock were examined. Exposure to either 40°C (P < 0.05) or 41°C (P < 0.001) increased the percentage of cells that were apoptotic compared those at 38.5°C (Fig. 3A). When embryos were subjected to 40°C, there was a quadratic (P < 0.01) effect of time on the percentage of cells that were apoptotic. The percentage of apoptosis was higher for embryos at 40°C than for embryos at 38.5°C, but the percentage of apoptosis at 40°C was similar for durations of 3, 6, and 9 h. The effect of time on the induction of apoptosis was even more pronounced at 41°C. Again, there was a quadratic (P < 0.001) increase in the percentage of apoptotic cells as time of exposure increased. Neither 40°C nor 41°C affected the total number of cells per embryo (Fig. 3B).
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Lack of Heat-Induced Apoptosis in Bovine Embryosat the 2- or 4-Cell Stage
Representative confocal digital images of embryos assessed by TUNEL for apoptosis 24 h after collection at the 2-cell stage are shown in Figure 1D (embryo at 38.5°C continuously) and Figure 1, E and F (embryos exposed to 41°C for 9 h and 38.5°C for 15 h, respectively, before TUNEL labeling). Note the absence of TUNEL labeling in all embryos and the increased incidence of nuclear fragmentation and reduced cell number in heat-shocked embryos.
When data were analyzed, exposure of 2- or 4-cell embryos to 41°C for 9 h had no effect on the percentage of cells undergoing apoptosis 24 h after initiation of heat shock compared with embryos cultured continuously at 38.5°C. Indeed, the proportion of apoptotic cells at these stages of embryonic development was very low and ranged from 0.04%–0.66% (Fig. 4A). This compares to 7.8%–8.0% apoptosis in 16-cell embryos collected at Day 5 and cultured at 38.5°C (Figs. 2A and 3A). Although heat shock did not affect apoptosis, at the 2-cell (38.5 vs. 41°C, P < 0.001) and 4-cell stages (38.5 vs. 41°C, P < 0.001), heat shock reduced total embryo cell number 24 h after initiation of treatment (Fig. 4B).
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Heat-Induced Apoptosis and Induced Thermotoleranceat the 8- to 16-Cell Stage
Two experiments were conducted to determine whether apoptosis occurs at the 8- to 16-cell stage. In the first study, exposure of Day 3 embryos at the 8- to 16-cell stage to 40°C or 41°C for 9 h had no effect on the percentage of apoptotic cells (Fig. 5A) or on total embryo cell number (Fig. 5B) at 24 h after initiation of temperature treatments. In the second experiment (Fig. 6), 8- to 16-cell embryos were collected on Day 4 rather than on Day 3. In addition, we tested whether exposure to a mild heat shock of 40°C for 80 min would make embryos more resistant to apoptosis induced by a severe heat shock of 41°C for 9 h. There was no difference between control embryos at 38.5°C and embryos subjected to 40°C for 80 min on the percentage of cells undergoing apoptosis or embryo cell number (Fig. 6A). In contrast, exposure to 41°C for 9 h increased (38.5 vs. 41°C, P < 0.001) the proportion of cells that were apoptotic 24 h after initiation of temperature treatments and reduced (38.5 vs. 41°C, P < 0.01) embryo cell number. Preincubation at 40°C for 80 min, however, blocked (40/41°C vs. 41°C, P <0.001) heat-induced apoptosis. Embryo cell number remained lower (38.5 vs. 40/41°C, P <0.001) for heat shocked embryos than controls, however, regardless of whether embryos were preincubated at 40°C (Fig. 6B).
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Caspase Activity
Representative digital images illustrating caspase activity in 2-cell embryos and embryos 16-cell stage are shown in Figure 2. Note the higher caspase activity for Day 5 embryos 16-cell stage exposed to heat shock of 41°C for 9 h (Fig. 7, D–F) compared with embryos exposed to 38.5°C (Fig. 7C). However, when 2-cell embryos were exposed to the same heat shock treatment there was no induction of caspase activity (Fig. 7, A and B).
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The effect of heat shock (41°C for 9 h) on caspase activity was dependent upon stage of development (stage x heat shock, P < 0.01). Heat shock increased (38.5 vs. 41°C, P < 0.001) caspase activity for Day 5 bovine embryos Fig. 8) compared with 38.5°C control. In contrast, caspase activity of 2-cell embryos was not affected by heat shock.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The temperatures that induced apoptosis included those characteristic of heat-stressed cows (40 and 41°C), suggesting that apoptosis could be induced in preimplantation embryos exposed to maternal hyperthermia. Moreover, the degree of apoptosis experienced by heat-shocked embryos generally reflected the severity of heat shock. The percent of cells that were positive for apoptosis increased with increasing heat shock temperatures and, at least at 41°C, increased with duration of heat shock. At 40°C, induction of apoptosis was also time dependent, with no increase in apoptosis seen after an exposure of 80 min (Fig. 6), but an increase in the percentage of apoptotic cells after 3, 6, or 9 h of exposure (Fig. 3). The observation that the degree of apoptosis following exposure to 40°C for 3, 6, and 9 h was generally similar could possibly reflect time-dependent cellular adaptation to prevent apoptosis following prolonged exposure to a mild heat shock of 40°C that was not possible for a more severe heat shock of 41°C.
Acquisition of heat-induced apoptosis is developmentally regulated and does not occur until approximately Day 4 after insemination at the 8- to 16-cell stage. Spontaneous apoptosis in bovine embryos is also first observed at the 8- to 16-cell stage [25]. The failure to observe apoptosis in 8- to 16-cell embryos collected at Day 3 after insemination may mean that either 1) development of apoptosis mechanisms are controlled by time as well as by the number of cleavage divisions, 2) that Day 4 embryos are more likely to have completed more cleavage divisions than Day 3 embryos, or 3) that Day 4 embryos include retarded embryos that are more susceptible to apoptosis.
Absence of heat-induced apoptosis in embryos heat shocked at the 2- and 4-cell stage is associated with a lack of activation of group II caspases. Failure of activation of these execution caspases is probably not a reflection of the absence of these enzymes or the absence of much of the signaling pathway for caspase activation. Experiments using the protein kinase C inhibitor staurosporine indicates that the cell death machinery is constitutively present in early cleavage embryos. Staurosporine could induce apoptosis in mouse embryos at the 1- to 4-cell stage [39, 40] and bovine embryos at the 1- to 16-cell stage [25]. Also, mRNA for caspase-2, -3, -6, and -12 are present throughout preimplantation embryonic development in the mouse, as are transcripts for the antiapoptotic Bcl-2, Bcl-xl, and Bcl-w and the proapoptotic Bax [40]. However, certain aspects of cell death are altered in early embryos because the time required for staurosporine to induce apoptosis in zygotes and 1- to 4-cell-stage embryos was longer than for blastocysts or other cell types [39]. One possibility is that antiapoptotic proteins are very high in early embryos and that heat-induced apoptosis becomes possible when amounts of these proteins decline during development.
It is also possible that the particular mechanisms for heat-induced apoptosis are dysfunctional in early cleavage-stage embryos. For heat shock and oxidative stress, induction of apoptosis is initiated upon activation of the enzyme sphingomyelinase, which hydrolyzes the membrane phospholipid sphingomyelin to generate the second-messenger ceramide [27–29]. Perhaps amounts of this enzyme or its substrate early in development make the early embryo unable to generate sufficient ceramide in response to heat shock for apoptosis to occur. Finally, that heat-induced apoptosis first occurs at a stage of development that is coincident with embryonic genome activation in the cow [1] means that it is possible that heat-induced apoptosis is dependent on transcriptionally controlled events in the embryo.
Previous exposure of Day 4 embryos at the 8- to 16-cell stage to a mild and transient heat shock of 40°C for 80 min blocked the induction of apoptosis induced by a subsequent severe heat shock of 41°C for 9 h. Such a phenomenon has been observed for others cells also [31–33]. The biochemical mechanisms by which mild heat shock prevents apoptosis induced by a more severe heat shock presumably involves HSP70 [16, 30, 41–44]. Besides protecting cells from heat shock, HSP70 can protect cells against several apoptotic stimuli, including DNA damage, UV irradiation, serum withdrawal, and chemotherapeutic agents [45]. The mechanism by which HSP70 exerts its antiapoptotic action is not completely understood, but it can block multiple points along the apoptotic pathway. HSP70 and HSP72 inhibit poly-(ADP-ribose) polymerase cleavage [30, 33]. HSP70 also blocks cytochrome c release from mitochondria [44], processing of inactive procaspase-3 [30], and SAPK/JNK activation [30]. In addition, disruption of murine heat shock factor 1 gene increased heat-induced apoptosis [31]. The bovine embryo can produce higher amounts of HSP70 in response to heat shock as early as the 2-cell stage [46–48].
Heat shock also compromised embryonic viability as determined by the total number of cells per embryo at 24 h after the initiation of heat shock. The deleterious effect of heat shock on embryo cell number depended upon the magnitude of heat shock and stage of embryonic development. Thus, heat shock at the 2- to 4-cell stage caused a larger reduction in embryo cell number than heat shock at the morula stage. This finding is in agreement with earlier studies in which heat shock caused a greater reduction in development when applied at the 2-cell stage than the morula stage [4, 5] or at Day 3 after fertilization than at Day 4 [13]. Therefore, those embryos that were at stages of development that were capable of heat-induced apoptosis were more resistant to the deleterious effect of heat shock on development. One possibility is that the sensitivity of the early embryo to heat shock is a reflection, at least in part, of the failure of the embryos to undergo heat-induced apoptosis to remove damaged cells from the embryonic lineage. There is some evidence that induction of limited apoptosis by heat shock does not necessarily inhibit embryonic development because embryos 16 cells that experienced apoptosis induced by 40 or 41°C did not have a reduced cell number 24 h after heat shock. Further studies evaluating other indices of development as well as the effects of inhibitors of apoptosis on development should add insight into the implications of stress-induced apoptosis for embryonic survival.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Research was supported in part by grants from the U.S. Department of Agriculture (TSTAR 95-34135-1860 and NRICGP 96-35205-3728), the Florida Milk Checkoff Program, and a fellowship to F.F.P.-L. from CAPES, a Brazilian research funding agency. This is journal series R-08343 of the Florida Agricultural Experiment Station. 
2 Correspondence: Peter J. Hansen, Department of Animal Sciences, University of Florida, P.O. Box 110910, Gainesville, FL 32611-0910. FAX: 352 392 5595; hansen{at}animal.ufl.edu
Accepted: November 14, 2001.
Received: September 5, 2001.
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R. M. Rivera, G. M Dahlgren, L. A. de Castro e Paula, R. T Kennedy, and P. J Hansen Actions of thermal stress in two-cell bovine embryos: oxygen metabolism, glutathione and ATP content, and the time-course of development Reproduction, July 1, 2004; 128(1): 33 - 42. [Abstract] [Full Text] [PDF]
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R. M. Rivera, K. L. Kelley, G. W. Erdos, and P. J. Hansen Reorganization of Microfilaments and Microtubules by Thermal Stress in Two-Cell Bovine Embryos Biol Reprod, June 1, 2004; 70(6): 1852 - 1862. [Abstract] [Full Text] [PDF]
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J. L. Tremoleda, T. A.E. Stout, I. Lagutina, G. Lazzari, M. M. Bevers, B. Colenbrander, and C. Galli Effects of In Vitro Production on Horse Embryo Morphology, Cytoskeletal Characteristics, and Blastocyst Capsule Formation Biol Reprod, December 1, 2003; 69(6): 1895 - 1906. [Abstract] [Full Text] [PDF]
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R. M. Rivera, K. L. Kelley, G. W. Erdos, and P. J. Hansen Alterations in Ultrastructural Morphology of Two-Cell Bovine Embryos Produced In Vitro and In Vivo Following a Physiologically Relevant Heat Shock Biol Reprod, December 1, 2003; 69(6): 2068 - 2077. [Abstract] [Full Text] [PDF]
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P. Fedorcsak and R. Storeng Effects of Leptin and Leukemia Inhibitory Factor on Preimplantation Development and STAT3 Signaling of Mouse Embryos In Vitro Biol Reprod, November 1, 2003; 69(5): 1531 - 1538. [Abstract] [Full Text] [PDF]
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J. O. Gjorret, H. M. Knijn, S. J. Dieleman, B. Avery, L.-I. Larsson, and P. Maddox-Hyttel Chronology of Apoptosis in Bovine Embryos Produced In Vivo and In Vitro Biol Reprod, October 1, 2003; 69(4): 1193 - 1200. [Abstract] [Full Text] [PDF]
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