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Developmental Changes in Inhibitory Effects of Arsenic and Heat Shock on Growth of Pre-Implantation Bovine Embryos

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MOLECULAR REPRODUCTION AND DEVELOPMENT 63: (2002) Developmental Changes in Inhibitory Effects of Arsenic and Heat Shock on Growth of Pre-Implantation Bovine Embryos C.E. KRININGER III, S.H. STEPHENS,
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MOLECULAR REPRODUCTION AND DEVELOPMENT 63: (2002) Developmental Changes in Inhibitory Effects of Arsenic and Heat Shock on Growth of Pre-Implantation Bovine Embryos C.E. KRININGER III, S.H. STEPHENS, AND P.J. HANSEN* Department of Animal Sciences, University of Florida, Gainesville, Florida ABSTRACT Although sensitive to various disrupters, pre-implantation embryos possess some cellular cytoprotective mechanisms that allow continued survival in the face of a deleterious environment. For stresses such as heat shock, embryonic resistance increases as development proceeds. Present objectives were to determine whether (1) arsenic compromises development of pre-implantation bovine embryos, (2) developmental changes in embryonic resistance to arsenic mimic those seen for resistance to heat shock, and (3) developmental patterns in induction of apoptosis by arsenic are correlated with similar changes in resistance of embryos to inhibitory effects of arsenic on development. Bovine embryos produced by in vitro fertilization were exposed at the two-cell stage or at day 5 after insemination (embryos 16- cells in number) to either sodium arsenite (0, 1, 5, or 10 mm) or heat shock (exposure to 418C for 0, 3, 4.5, 6, or 9 hr). Arsenic induced apoptosis and increased group 2 caspase activity for embryos at the 16-cell stage, but not for embryos at the two-cell stage. In contrast to these developmental changes in apoptosis responses, exposure to arsenic reduced cell number 24 hr after exposure for both two-cell embryos and embryos 16-cells. Similarly, the percentage of embryos that developed to the blastocyst stage at day 8 after fertilization was reduced by arsenic exposure at both stages of development. Heat shock, conversely, reduced development to the blastocyst stage when applied at the two-cell stage, but not when applied to embryos 16-cells at day 5 after insemination. In conclusion, arsenic can compromise development of bovine pre-implantation embryos, the temporal window of sensitivity of embryos to arsenic is wider than for heat shock, and cellular cytoprotective responses that embryos acquire for thermal resistance are not sufficient to cause increased embryonic resistance to arsenic exposure. It is likely that despite common cellular pathologies caused by arsenic and heat shock, arsenic acts to reduce development in part through biochemical pathways not activated by heat shock. Moreover, the embryo does not acquire significant resistance to these perturbations within the time frame in development examined. Mol. Reprod. Dev. 63: , ß 2002 Wiley-Liss, Inc. ß 2002 WILEY-LISS, INC. Key Words: embryo; bovine; heat shock; arsenic; apoptosis INTRODUCTION Successful development of the pre-implantation embryo is dependent on maintenance of cellular function in the face of adverse conditions in the embryonic microenvironment. Embryonic resistance to certain disrupters increases as the embryo advances in development. For example, more advanced pre-implantation embryos are more resistant to heat shock (Edwards and Hansen, 1997; Ju et al., 1999), cyanide (Donnay et al., 2000), and bichlorinated biphenyls (Küchenhoff et al., 1999) than are less advanced embryos. Such a finding suggests that embryos acquire additional or more effective cytoprotective mechanisms as they proceed through development. In contrast, sensitivity of embryos to hydrogen peroxide changes little during the period of pre-implantation development (Morales et al., 1999) and embryonic sensitivity to cadmium (De et al., 1993) and chlorambucil (Giavini et al., 1984) increases as embryos advance through pre-implantation development. One developmentally-regulated process that may be involved in determining embryonic resistance to stress is apoptosis. Apoptosis may play an important role in protecting the embryo from embryotoxic conditions by removing damaged cells that would otherwise become necrotic or lead to the formation of dysfunctional daughter cells. Indeed, cells exposed to stress that cannot undergo apoptosis frequently undergo cell death more reminiscent of necrosis (Xiang et al., 1996; Woo et al., 1998). Evidence from bovine embryos exposed to Grant sponsor: USDA IFAFS program (USDA-CSREES); Grant number: ; Grant sponsor: Florida Milk Checkoff Program. S.H. Stephens s present address is Department of Wildlife and Fisheries Sciences, Texas A&M University, College Station, TX and Department of Marine Biology, Texas A&M University at Galveston, Galveston, TX *Correspondence to: P.J. Hansen, PO Box , Gainesville, FL Received 1 April 2002; Accepted 29 May 2002 Published online in Wiley InterScience (www.interscience.wiley.com). DOI /mrd.90017 336 C.E. KRININGER III ET AL. heat shock indicates that stress-induced apoptosis is a developmentally-regulated phenomenon. In particular, heat shock was not effective in induced apoptosis in bovine pre-implantation embryos until day 4 of development (Paula-Lopes and Hansen, 2002a). Acquisition of heat-induced apoptosis is temporally associated with developmental resistance of bovine embryos to heat shock (Edwards and Hansen, 1997; Ju et al., 1999) suggesting that apoptosis may be one of the mechanisms by which the embryo gains thermal tolerance. Moreover, inhibition of apoptosis responses increased sensitivity of bovine embryos to heat shock (Paula-Lopes and Hansen, 2002b). It is not known whether developmental acquisition of tolerance to other adverse conditions that can compromise embryonic survival is related to induction of apoptosis. One molecule that can compromise preimplantation embryonic development, at least in the mouse, is arsenic (Müller et al., 1986; Dix et al., 1998). The objectives of the present study were to determine whether (1) arsenic compromises development of pre-implantation bovine embryos, (2) developmental changes in embryonic resistance to arsenic mimic those seen for resistance to heat shock, and (3) developmental patterns in induction of apoptosis by arsenic are correlated with similar changes in resistance of embryos to inhibitory effects of arsenic on development. MATERIALS AND METHODS Materials Sodium arsenite was obtained from Sigma (St. Louis, MO). The terminal deoxynucleotidyl transferasemediated dutp nick end labeling (TUNEL) assay was performed with the In Situ Cell Death Detection Kit with fluorescein label purchased from Roche (Indianapolis, IN). Propidium iodide was obtained from Sigma. Polyvinylpyrrolidone (PVP) was purchased from Eastman Kodak (Rochester, NY), Prolong Antifade Kit was obtained from Molecular Probes (Eugene, Oregon), RQ1 RNA-free DNase was from Promega (Madison, WI) and RNase A was from Qiagen (Valencia, CA). The fluorogenic caspase substrate PhiPhiLux-G 1 D 2 was obtained from OncoImmunin, Inc. (Gaithersburg, MD). Materials for production and culture of embryos were obtained as described by Rivera and Hansen (2001). Oocyte Maturation, Fertilization, and Embryo Culture Embryos were produced using oocytes recovered from ovaries obtained at a local abattoir. Procedures for in vitro maturation of oocytes, in vitro fertilization, and embryo culture were performed as described by Rivera and Hansen (2001). After fertilization, putative zygotes were cultured in 50-ml drops of modified Potassium Simplex Optimized Medium (KSOM) overlaid with mineral oil. The KSOM, which contains 1 mg/ml BSA, was modified on the day of use by adding 2 3 mg/ml essentially fatty acid-free BSA, 2.5 mg/ml gentamicin, 1 essential amino acids (Basal Medium Eagle), and 1 non-essential amino acids (Minimal Essential Medium). Embryos were harvested from culture drops at the twocell stage (26 34 hr after insemination of cumulus oocyte complexes) or at the 16-cell stage (day 5 after insemination). Embryos were placed into fresh microdrops of modified KSOM. Embryos were randomly distributed in approximately equal numbers to drops for each treatment. Thus, while the number of embryos varied from 6 to 19 embryos per drop for two-cell embryos and 2 9 embryos per drop for 16-cell embryos, the number of embryos per drop was similar for each treatment within a replicate. Variations in the number of embryos per treatment reflect variation in the number of drops per replicate rather than in embryo number per drop. Drop volume was 50 ml for experiments on apoptosis and 25 ml for experiments where development was the endpoint. Effect of Arsenic on Apoptosis and Development 24 hr After Exposure to Arsenic Embryos were harvested at the two- and 16-cell stage and placed in fresh microdrops of modified KSOM medium containing 0, 1, 5, or 10 mm sodium arsenite. After 12 hr at 38.58C, embryos were washed in HEPES- TALP and returned to culture in fresh microdrops of modified KSOM for 12 hr at 38.58C. Embryos were then washed three times in 100-ml drops of 10 mm potassium phosphate, ph 7.4 containing 0.9% (w/v) NaCl, and 1 mg/ ml PVP (PBS/PVP), and fixed for 1 hr at room temperature in a 100-ml drops of 4% (w/v) paraformaldehyde and 0.9% (w/v) NaCl in 100-mM potassium phosphate, ph 7.4. Embryos were then washed three times in PBS/PVP and stored in this buffer at 48C for up to 30 days prior to evaluation for fragmented DNA using the TUNEL procedure. The TUNEL reaction was performed at room temperature using embryos placed in groups in individual wells of polystyrene, flat-bottom 96-well microtiter plate (Falcon, Becton Dickinson, Lincoln Park, NJ). After warming embryos for 30 min at room temperature, embryos were washed two times by transferring to wells containing 75-ml PBS/PVP for 6 min. Permeabilization of cellular membranes was accomplished by transfer of embryos to wells containing 75-ml permeabilization solution (0.5% (v/v) Triton X-100, 0.1% (w/v) sodium citrate) for 30 min at room temperature. Embryos were washed twice and positive and negative control embryos were then incubated with DNase (50 U/ml) for 1 hr and washed twice in PBS/PVP. All embryos were transferred to wells containing 50 ml of TUNEL reaction mixture (prepared as provided by the manufacturer) and incubated for 1 hr in the dark. Negative controls were incubated in TUNEL reaction mixture in which the terminal deoxynucleotidyl transferase was excluded. After washing, embryos were incubated with RNase A (50 mg/ml in PBS/PVP) for 1 hr at room temperature in the dark, washed again and then transferred to wells containing propidium iodide (0.5 mg/ml in PBS/PVP) for 25 min at room temperature in the dark. Embryos were washed two more times in PBS/PVP. Embryos were transferred in a minimal amount of PBS/PVP to a clean ARSENIC, HEAT SHOCK, & EMBRYONIC DEVELOPMENT 337 microscope slide, covered with 15 ml Prolong 1 Antifade mounting medium, dried for min at 398C in a drying oven and then examined for total cell number (red and yellow nuclei) and apoptotic nuclei (yellow nuclei) using a Zeiss Axioplan 2 fluorescence microscope with dual filter. The experiment was replicated on three occasions with a total of embryos per group. Effect of Arsenic on Caspase Activity Caspase activity was measured using the fluorogenic substrate PhiPhiLux-G 1 D 2. This molecule, which produces increased green fluorescence upon cleavage, is specific for group II caspases, including caspase 3, 2, and 7. Embryos were harvested from microdrops at the twoand 16-cell stages. Embryos were placed in fresh microdrops of modified KSOM or modified KSOM containing 10 mm sodium arsenite. After 12 hr at 38.58C, embryos were washed three times in 50-ml drops of HEPES-TALP and transferred to 25-ml microdrops containing modified KSOM (negative control) or modified KSOM containing 5 mm PhiPhiLux-G 1 D 2 (PhiPhi; OncoImmunin). Embryos were incubated in a humidified box in an oven at 398C for min, removed from drops, washed once in 50 ml HEPES-TALP and then transferred in a small volume to slide glass microscope slides. Embryos were covered with a cover slip and examined within 30 min for fluorescence using a Zeiss Axioplan 2 fluorescence microscope. Digital images were obtained using a Spot camera (Diagnostic Instruments, Sterling Heights, MI) and software and stored as.tiff files. A total of 10 embryos per group collected in one replicate were examined. Effect of Arsenic on Development to the Blastocyst Stage Embryos were harvested at the two- and 16-cell stage and placed in fresh microdrops of modified KSOM medium containing 0, 1, 5, or 10 mm sodium arsenite. After 12 hr at 38.58C, embryos were washed in HEPES- TALP and returned to culture in fresh microdrops of modified KSOM. The proportion of embryos developing to blastocyst was measured on day 8 after insemination. The experiment was replicated on a total of five occasions (two-cell; embryos per group) or nine occasions ( 16-cell; embryos per group). Effect of Heat Shock on Development to the Blastocyst Stage Embryos were harvested at the two- and 16-cell stage, placed in fresh microdrops of modified KSOM medium, and then either cultured at 38.58C and 5% (v/v) CO 2 continuously or placed in an incubator at 418C and 7% (v/v) CO 2 for 3, 4.5, 6, or 9 hr. The increase in CO 2 content at 418C was made to account for the lower solubility of CO 2 at higher temperature and to assure that ph remained constant in the face of changing temperature (Rivera and Hansen, 2001). At the end of the heat shock period, embryos were returned to an incubator at 38.58C and 5% (v/v) CO 2. The proportion of embryos developing to blastocyst was measured on day 8 after insemination. The experiment was replicated on a total of eight occasions (two-cell; embryos per group) or 10 occasions ( 16-cell; embryos per group). Statistical Analysis Data were analyzed by least-squares analysis of variance using the GLM procedure of SAS (SAS, 1989). For experiments evaluating development, the percentage of embryos that developed to the blastocyst was calculated for each replicate. Data on the percent of blastomeres positive for TUNEL reaction were calculated on a per embryo basis. Percentage data were subjected to an arcsin transformation before analysis to normalize data. Probability values reported in the article are based on analysis of transformed data, while least-squares means are from analysis of untransformed data. Mathematic models for analysis of variance included effects of treatment (e.g., arsenic concentration), replicate, and treatment replicate. Some analyses were also done to examine effects of stage of exposure and stage treatment on development to the blastocyst stage. Effects of arsenic concentration were separated into individual degree of freedom contrasts to determine linear, quadratic, and cubic effects of arsenic concentration. The polynomial coefficients to perform this analysis were calculated using the PROC IML procedure of SAS. In all analyses, replicate was considered a random effect, while other main effects were considered fixed. Therefore, treatment replicate was used as the error term to test effect of treatment. RESULTS Effects of Arsenic on Two-Cell Embryos To determine whether arsenic exposure could induce apoptosis, two-cell embryos were cultured for 12 hr with various concentrations of arsenic and the percent of blastomeres positive for the TUNEL reaction determined at 24 hr after arsenic exposure (i.e., at 12 hr after the end of arsenic treatment). The proportion of two-cell embryos exhibiting apoptosis in the absence of arsenic exposure was very low in fact, only 1 of 27 embryos exhibited apoptosis in the absence of arsenic exposure. Treatment of embryos with arsenic did not increase the percentage of cells labeled with the TUNEL reagent (Fig. 1). Arsenic also did not increase group 2 caspase activity of two-cell embryos as determined by cleavage of the substrate PhiPhiLux-G 1 D 2 and generation of green fluorescence (Fig. 2C). There was little or no detectable caspase activity in control or arsenic-treated embryos. Effects of arsenic on developmental competence of embryos were ascertained in two ways. First, cell number at 24 hr after initiation of a 12-hr arsenic exposure was determined. Cell number was affected by concentration of arsenic in a dose response relationship best described as linear (P 0.001) (Fig. 1). In the second experiment, development to blastocyst was determined for embryos exposed to arsenic for 12 hr at the two-cell stage. Again, the proportion of embryos developing to 338 C.E. KRININGER III ET AL. Fig. 1. Developmental changes in effects of arsenic on embryonic growth and induction of apoptosis. Embryos were collected at the twocell stage or on day 5 after insemination (embryos 16 cells) and exposed to various concentrations of arsenic for 12 hr. At 24 hr after initiation of arsenic exposure, embryos were stained using propidium iodide and the TUNEL procedure to determine the cell number and degree of apoptosis. The experiment was replicated on three occasions with a total of embryos per group. Results are least-squares meanssem. the blastocyst stage was reduced by arsenic treatment in a linear manner (linear contrast, P 0.01) (Fig. 3). Effect of Arsenic on Embryos 16 Cells at Day 5 After Insemination In contrast to two-cell embryos, apoptotic cells were observed in the absence of arsenic exposure (9.9% of blastomeres were positive for the TUNEL reaction). Culture of embryos with arsenic increased the proportion of blastomeres that exhibited apoptosis in a concentration-dependent manner best described as a linear response (P ¼ 0.056) (Fig. 1). Exposure to 10-mM arsenite also increased group 2 caspase activity as determined by cleavage of PhiPhiLux-G 1 D 2 (compare Fig. 2A for embryo without arsenic with Fig. 2B for embryo with arsenic). As shown in Figure 1, cell number at 24 hr after initiation of a 12-hr arsenic exposure was reduced by increasing concentrations of arsenic in a cubic manner (P 0.05). The cubic nature of the response reflects the fact that the reduction in cell number caused by arsenic exposure was similar for all concentrations of arsenic. In the second experiment, development to blastocyst was determined for embryos exposed to arsenic for 12 hr. The proportion of embryos developing to the blastocyst stage was reduced by arsenic treatment in a linear manner (P 0.001) (Fig. 3). A combined set of blastocyst development data from exposure at the two- and 16-cell stage was also analyzed. In this case, there was an effect of arsenic concentration on development (P 0.05), but there was no concentration stage of development interaction. Such a result indicates arsenic exposure affected development to the blastocyst stage equally at both embryonic stages. Heat Shock Effects on Embryonic Development To compare developmental changes in resistance to arsenic to those with heat shock, an experiment was performed in which development to blastocyst was determined for embryos exposed to heat shock at the two-cell stage or for embryos 16-cells (Fig. 4). Application of heat shock at the two-cell stage reduced the proportion of embryos that developed to the blastocyst stage (P 0.001). In contrast, application of heat shock to embryos 16-cells at day 5 after fertilization did not affect the proportion of embryos that became blastocysts. When a combined data set was analyzed, there was a stage of exposure temperature interaction (P ¼ 0.05), indicating that effects of heat shock were Fig. 2. Group 2 caspase activity in bovine embryos. Represented are an embryo 16-cells collected at day 5 post IVF (A), an arsenic-treated embryo 16-cells collected at day 5 (B) and a two-cell embryo treated with arsenic (C). Caspase activity was monitored by a fluorogenic substrate for group 2 caspases. Note that for the control day 5 embryo, only a few cells stained intensely for caspase, while there was intense reaction product over more than 50% of the arsenic-treated day 5 embryo. There was no detectable caspase activity for the arsenictreated two-cell embryo. ARSENIC, HEAT SHOCK, & EMBRYONIC DEVELOPMENT 339 Fig. 3. Develop
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