Evidence That Glutathione Is Involved in Thermotolerance of ...

BIOLOGY OF REPRODUCTION 52, 1296-1301 (1995)

Evidence That Glutathione Is Involved in Thermotolerance of Preimplantation Murine Embryos' 2 C.F. ARECHIGA, A.D. EALY, 3 and PJ. HANSEN

Departmentof Dairy and Poultry Sciences, University of Florida, Gainesville, Florida 32611-0920 ABSTRACT Experiments were conducted to determine whether or not glutathione (GSH) is involved in thermotolerance responses of murine morulae. In the first experiment, morulae were exposed to either homeothermic temperature (37'C), mild heat shock 0 (40°C for 1 h), severe heat shock (43 C for 2 h), or a mild heat shock followed by severe heat shock (to induce thermotolerance). Exposure to mild heat shock did not affect viability and development, but severe heat shock reduced viability (i.e., live/dead 0 staining) and the proportion of morulae that developed to blastocysts. This effect of 43 C was reduced if embryos were first 0 exposed to a mild heat shock of 40 C. In the presence of DL-buthionine-[S,R]-sulfoximine (BSO), an inhibitor of GSH synthesis, the ability of 40°C to confer thermotolerance was reduced. BSO decreased embryonic GSH content but did not decrease overall protein synthesis. In another experiment, administration of S-adenosyl-L-methionine, an inducer of GSH synthesis, decreased the 0 deleterious effects of heat shock of 43 C for 2 h on viability and percentage of embryos that became blastocysts. Addition of 5 of 42'C for 2 h on viability but not on continued development. The results suggest a effect the reduced ester or GSH M GSH 1 role for GSH-dependent mechanisms in the processes by which murine embryos limit deleterious effects of heat shock.

INTRODUCTION The early mammalian embryo is compromised by exposure to elevated temperatures; maternal heat stress during early stages of development causes a decrease in embryonic survival [1-3], and exposure of embryos to elevated temperature in culture (i.e., heat shock) reduces viability and subsequent development [4-8]. As for most cells, mouse embryos can be made resistant to heat shock through a process termed induced thermotolerance whereby cells can resist a severe heat shock if first exposed to a mild heat shock [7, 8]. The process of induced thermotolerance first occurs at the 8-cell stage for embryos cultured in vitro from the 2-cell stage, and at the blastocyst stage for embryos collected from the reproductive tract [8]. Induction of thermotolerance in most cells is closely associated with synthesis of heat shock proteins (HSP) [9, 10]. In fact, induction of thermotolerance in fibroblasts is blocked by intracellular administration of antibodies to heat shock protein 70 (HSP70) [11], while thermotolerance can be induced in oocytes by intracellular injection of HSP70 mRNA [12] and in fibroblasts by transfection of the HSP70 gene [13]. The synthesis of HSPs is not the only prerequisite for induced thermotolerance, however. In particular, the antioxidant tripeptide, glutathione (GSH), has been implicated Accepted January 31, 1995. Received September 17, 1993. 'Research supported by the Florida Dairy Checkoff Program and USDA-CBAG Grant No. 9204572. This is Journal Series No. R-03389 of the Florida Agricultural Experiment Station. C.FA is supported by the CONACYF/IIE Grants Program (Mexico) and CIBMYC (International Center for Cell and Molecular Biology), AC., Monterrey, N.L., M6xico. 'Correspondence: Dr. PJ. Hansen, Department of Dairy and Poultry Sciences, University of Florida, P.O. Box 110920, Gainesville, FL 32611-0920. FAX: (904) 392-

5595. 3Current address: Department of Animal Sciences, University of Missouri, Columbia, MO 65211.

as being critical for thermotolerance. Intracellular concentrations of GSH have been reported to increase after exposure to heat shock in several cells [14-17]. Inhibition of GSH synthesis with DL-buthionine-[S,R]-sulfoximine (BSO) blocked induced thermotolerance in hamster fibroblasts [14,18,19] and rat postimplantation embryos [17]. Supplementation of medium with GSH increased resistance of bovine embryos [20] to heat shock. It is likely that GSH acts during heat shock to limit the effects of free radicals generated during heat shock [21]. GSH might also be essential for the acquisition of thermotolerance by allowing for increased synthesis of HSPs [18], although this has been questioned [17]. Taken together, these results indicate that GSH could play a critical role in thermotolerance responses. Accordingly, a series of experiments was conducted to better clarify the role of GSH in induction of thermotolerance of murine embryos. One question examined was whether inhibition of GSH synthesis blocked induction of thermotolerance and whether stimulation of GSH synthesis increased resistance of embryos to heat shock. An additional question examined was whether administration of GSH and GSHrelated molecules exerted a thermoprotective effect on embryos. MATERIALS AND METHODS Materials Silicon oil was purchased from Aldrich Chemical Co. (Milwaukee, WI). [3 5 S]Cysteine (> 600 Ci/mol) and [3 5 S]methionine (> 1000' Ci/mmol) were purchased from Amersham (Arlington Heights, IL). Equine CG, hCG, heattreated fetal calf serum (htFCS), 4',6'-diamidino-2-phenylindole (DAPI), BSO, S-adenosyl-L-methionine (SAM), GSH (reduced form), GSH reductase, p-nicotinamide adenine 1296

ROLE OF GSH IN INDUCED THERMOTOLERANCE dinucleotide phosphate (reduced form; NADPH), 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB), and other reagents were purchased from Sigma Chemical Co. (St. Louis, MO). The monoethyl ester of GSH was synthesized through use of the sulfuric acid esterification procedure described by Anderson and Meister [22]. The synthesized GSH ester was confirmed to be > 90% pure by thin-layer chromatography using MK6F plates (Whatman, Hillsboro, OR), a solvent of n-propanol:acetic acid:water (16:3:5 [v/v]), and ninhydrin (Whatman, Hillsboro, OR) as a detection reagent [22]. The M2 and M16 media were prepared as described by Hogan et al. [23], and CZB medium was prepared as described by Chatot et al. [24].

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Time (hours) Superovulation, Embryo Collection, and Culture Mice of the ICR outbred strain (Harlan Sprague Dawley Inc., Indianapolis, IN) and B6D2F1/J hybrid strain (The Jackson Laboratory, Bar Harbor, ME) were housed at 25°C in 14L:10D photoperiodic cycles with 2400 h as the midpoint of the dark cycle. Prepubertal female mice, 21-25 days of age (ICR), and postpubertal female mice, 42-56 days of age (B6D2F1/J), were superovulated with 5 to 10 IU eCG, i.p., followed 44-48 h later by 5 to 7.5 IU of hCG, i.p. Females were then placed with males overnight, and coitus was verified the next morning by the presence of a vaginal plug. At 24 h after detection of plugs, females were killed and the oviducts collected. Embryos were retrieved by flushing oviducts with M2 medium supplemented with 0.4% (w/v) BSA. Embryos were cultured until the morula stage in groups of 5-10 in 5-7-1l microdrops of M16 containing 0.4% BSA covered with twice-extracted silicon oil at 37°C in an atmosphere of 5% CO 2 in humid air. Morulae were transferred in groups of 5-10 into 5-pl microdrops of M16 medium containing 10% (v/v) htFCS and were exposed to various treatments. For each day of embryo collection, embryos were randomly assigned to treatment without reference to donor. Determinationof Viability and Development At the end of culture, viability was determined by use of the fluorescent compound, DAPI [25]. Embryos were placed in Dulbecco's PBS (DPBS) containing 0.0001% (w/v) DAPI. After 15-20 min, embryos were evaluated for fluorescing nuclei through use of an epifluorescent microscope with ultraviolet excitation and a 490-nm emission filter. Embryos were considered live if less than approximately one third of the cells fluoresced and dead if more than one third fluoresced. Stage of embryonic development was determined by visual observation during DAPI staining. Embryos were classified as having progressed in development if they advanced from the morula to the blastocyst stage of development.

FIG. 1. Schematic diagram of experimental design used to test the effect of BSO on induction of thermotolerance. Exposure to 43°C reduces embryonic viability (as determined by live/dead staining) and continued development. It was anticipated that previous exposure to 40°C would induce embryonic thermotolerance and thereby reduce effects of a subsequent exposure to 43°C (40/43°C group). When present, BSO was added through a period equivalent to the end of the 40°C treatment to block GSH synthesis during induction of thermotolerance. Gray shading represents 37 C.

Effect of BSO on Induction of Thermotolerance A schematic diagram of the experimental design is presented in Figure 1. At hour 4, morula-stage embryos were exposed to one of four temperature treatments for 5 h (n = 40-56 per group). Temperature treatments were as follows: 37°C continuously; 40°C from hour 4 to 5 (40°C for 1 h; mild heat shock); 43°C from hour 7 to 9 (43°C for 2 h; lethal heat shock); and 40°C from hour 4 to 5 and 43°C from hour 7 to 9 (40°C/43°C; temperature treatment to induce thermotolerance). For some groups, 1 mM BSO was present from hour 0 to 5 (i.e., until a time equivalent to the end of the 40°C treatment; total duration of BSO treatment = 5 h). All embryos were transferred into fresh medium without BSO at hour 5 and cultured at 37 0C. Viability was assessed by DAPI staining at hour 23. Developmental status was evaluated at hour 23 (percentage becoming blastocysts) and hour 36 (percentage undergoing hatching). Effects of Heat Shock and BSO on GSH Content Morulae were cultured at 37 0C in groups of 5-10 in microdrops of M16 medium containing 10% htFCS with or without 1.0 mM BSO for 5 h. Embryos were then either maintained at 37 0C for 3 h or transferred to 41°C for 1 h followed by 37°C for 2 h. Embryos were then frozen in 5 IlI distilled water until analysis. Subsequently, embryos were thawed, pooled into groups of 10-17 embryos, mixed with a volume of 1.25 M H3 PO4 equal to the volume of embryos, and assayed for GSH through the use of a modified DTNBGSSG reductase recycling assay [26]. The limit of detection was 0.14 pmol. A total of three groups of embryos were assayed for each treatment.

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AREECHIGA ET AL. TABLE 1. Effect of BSO on induction of thermotolerance in murine morulae. Temp

BSO'

No. of embryos

%Live at 23 hb

% Blastocyst at c 23 h

%Hatched at 36 hd

-

37°C 40°C 43°C

52 52 55

100 90 7

92 94 20

44 52 5

-

40/43C

53

74

45

26

5h 5h 5h 5h

37°C 40C 43°C 40/43°C

52 40 41 56

90 88 0 25

92 73 27 30

37 23 5 5

aBSO was added for 5 h (until the end of 40°C treatment). bViability was affected by BSO, temperature, and BSO x temperature (p < 0.001). CPercent development was affected by BSO (p < 0.05), temperature (p < 0.001), and BSO x temperature (p < 0.07). 'Percent hatched was affected by BSO (p < 0.001), temperature (p < 0.001), and BSO x temperature (p < 0.06).

Effect of Heat Shock and BSO on Protein Synthesis Morula-stage embryos were placed in groups of 5-10 in microdrops of M16 medium containing 10% htFCS with or without 1.0 mM BSO for 5 h. During this time, embryos were cultured at either 370C continuously or at 37°C from hour 0 to 4 followed by 40°C from hour 4 to 5. Embryos were then washed in CZB medium containing 1% (v/v) polyvinyl alcohol and cultured for 4 h at 37°C in groups of 5-10 in microdrops of 50 z 1 CZB medium containing 1% polyvinyl alcohol, 1 mCi/ml [3 5S]cysteine, and 1 mCi/ml [3 5S]methionine. Embryos were next washed in cold CZB medium and transferred individually in a minimum volume of CZB medium (-2 1) to a microfuge tube containing 30 l1100 mM Tris (pH 8.3), 2 mM EDTA, 0.5% (w/v) sodium deoxycholate, 0.5% (w/v) Nonidet P-40, 0.1% (w/v) SDS, 1 mg/ml ovalbumin, and 1% (v/v) -mercaptoethanol. Samples were boiled for 2-3 min and placed at -70°C un-

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Effect of S-adenosyl-L-methionine (SAM) on Responses to Heat Shock Morulae (53-70 per treatment) were cultured for 15 h (i.e., hour 0 to 15) in M16 medium containing 10% htFCS with or without 100 pIM SAM, a molecule that stimulates synthesis of GSH [28,29]. Both groups of embryos were then transferred to fresh medium without SAM and exposed to 37°C continuously or to 43°C from hour 17 to 19. Embryos were assessed for viability and development to blastocysts at hour 27 and for hatching at hour 36. Effect of Reduced GSH and Monoethyl GSH Ester on Responses to Heat Shock Morulae were cultured for 24 h (i.e., hour 0 to 24) in M16 medium containing 10% htFCS that either was not supplemented or was modified to contain 5 ,IM reduced GSH or 5 p.M monoethyl GSH ester. Embryos were exposed to 37°C continuously (i.e., hour 0 to 24) or to 42°C for 2 h (i.e., 37°C from hour 0 to 2; 42°C from hour 2 to 4; and 37°C from hour 4 to 24). Embryos were then assessed for viability and development to blastocysts at hour 24.

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FIG. 2. Effect of BSO and heat shock on GSH content of mouse morulae. Content was affected by BSO treatment (p < 0.04).

Data were subjected to analysis of variance using the General Linear Models procedure of SAS [30]. Analysis of variance was utilized even for categorical data for reasons outlined by Wilcox et al. [31]. Except for experiments on GSH content, in which pools of embryos were assayed, one embryo was considered as the experimental unit. All analyses were originally performed with experiment (i.e., day of embryo collection) and interactions with experiment in the model. These terms were sometimes removed in subsequent analyses if they did not account for a large proportion of the variation.

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ROLE OF GSH IN INDUCED THERMOTOLERANCE TABLE 2. Effect of SAM on responses of morulae to heat shock. Temperature

No. of embryos

%Livea

%Developed to blastocystb

%Hatched'

-

37°C 43°C

54 53

98 25

76 38

31 28

+ +

37°C 43°C

64 70

95 79

83 51

31 19

SAM

'Effect of temperature, SAM, and temperature x SAM (p < 0.001). bEffect of temperature (p < 0.001), and SAM (p < 0.1). 'Effect of temperature (p < 0.05).

For effects of BSO, the model included main effects of temperature (37, 40, 43, 40/43°C) and BSO in a 2x4 factorial design. Effects of BSO on GSH content and protein synthesis were analyzed using effects of temperature (37°C vs. 40°C), dosage of BSO (0 vs. 1 mM), and the interaction. To determine effects of SAM, data were analyzed utilizing a 2 x 2 factorial design with effects of SAM, temperature, and the interaction. Effects of GSH and monoethyl GSH ester were analyzed in a 3x2 factorial design to determine effects of treatment (control, GSH, GSH ester) in embryos exposed to two different temperatures (37 0C vs. 420C). Effects of treatment and treatment x temperature were separated into individual degree-of-freedom comparisons using orthogonal comparisons (control vs. GSH + GSH ester; GSH vs. GSH ester).

at 410C also reduced GSH content of morulae not treated with BSO by 46%, but the temperature x treatment interaction effect was not significant. Effect of BSO on Protein Synthesis Rather than inhibiting protein synthesis by embryos, BSO tended to increase (p < 0.1) incorporation of [35 S]methionine and [35S]cysteine into TCA-precipitable protein at 37°C (least squares means = 2035 + 617 vs. 3183 ± 477 dpm for control and 1 mM BSO, respectively) and 40°C (1103 + 552 vs. 1642 + 537 dpm). Exposure to 40°C reduced protein synthesis (p < 0.02), but there was no temperature x BSO interaction. Effect of SAM on Resistance to Heat Shock

RESULTS Effect of BSO on Induction of Thermotolerance Results are shown in Table 1. There were BSO x temperature interactions for viability (p < 0.001) and for the proportion of embryos developing to blastocysts (p < 0.07) and undergoing hatching (p < 0.06). These interactions are attributable to BSO's having reduced the ability of 40°C to block deleterious effects of 43°C. In the absence of BSO, 40°C did not decrease viability or development. Moreover, embryos that were first exposed to 40°C were less affected by subsequent exposure to 43°C. For example, while only 7% of embryos exposed to 430C were alive at 23 h, this percentage was increased to 74% if embryos were first exposed to 40 0C. The induction of thermotolerance by 40°C was also apparent, though of lower magnitude, for percentage development to the blastocyst and hatched blastocyst stage. Preincubation with BSO reduced or blocked the induction of thermotolerance by 40°C. For example, the percentage of live embryos was 0% for those exposed to 43°C and was 25% for those exposed to 40/43°C. Effects of Heat Shock and BSO on Embryonic GSH Concentrations As shown in Figure 2, BSO caused a large decrease in GSH content (p < 0.04). Amounts of GSH were undetectable in four of six pools of BSO-treated embryos. Heat shock

As shown in Table 2, exposure to 43°C reduced the percentage of embryos alive at 27 h (p < 0.001) and the percentage that became blastocysts (p < 0.001) and underwent hatching (p < 0.05). There was a SAM x temperature interaction affecting viability (p < 0.001), because the deleterious effects of 43°C were reduced in embryos treated with SAM. Treatment with SAM tended (p < 0.10) to increase the proportion of embryos that became blastocysts at both 37°C and 43°C. There was no temperature x treatment interaction.

TABLE 3. Effect of GSH and monoethyl GSH ester on responses of morulae to heat shock. Temperature

No. of embryos

%Livea

% Developed to blastocystb

None

37°C 42°C

88 92

90.9 35.9

76.1 29.4

GSH (5 M)

37°C 42'C

94 100

90.4 58.0

83.0 36.0

GSH ester (5 pLM)

37°C 42'C

95 103

95.8 47.6

88.4 24.3

Treatment

aEffect of temperature (p < 0.001), treatment (p < 0.02), and temperature

x treatment (p < 0.02); significant orthogonal contrasts were control vs. GSH + GSH ester (p < 0.01), temperature x control vs. GSH + GSH ester (p < 0.04), and temperature x GSH vs. GSH ester (p < 0.04). bEffect of temperature (p < 0.001) and temperature x treatment (p < 0.04); significant orthogonal.contrasts were control vs. GSH + GSH ester (p = 0.07) and temperature x GSH vs. GSH ester (p < 0.03).

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ARECHIGA ET AL.

Effect of GSH and Monoetbyl GSH Ester on Resistance to Heat Shock

Results are shown in Table 3. Exposure to 42°C reduced the percentage of embryos alive at 24 h (p < 0.001) and the percentage that became blastocysts (p < 0.001). There was a treatment x temperature interaction affecting viability (p < 0.02), because the deleterious effects of 42°C were reduced in embryos treated with either GSH or GSH ester. GSH was more effective than GSH ester at increasing viability at 42°C as indicated by the GSH vs. GSH ester x temperature interaction (p < 0.04). Development to blastocysts was affected by a temperature x treatment interaction. The interaction was attributed to the fact that both GSH and GSH ester increased development at 37°C slightly whereas only GSH caused a slight increase in development at 40°C. DISCUSSION

The model used in the present study to examine the induction of thermotolerance in preimplantation embryos was originally described by Ealy and Hansen [8]. In the original experiments [8], exposure to 40°C reduced the effects of 43°C for 2 h on viability but not on development. Thus, it was more difficult to block effects of severe heat shock on development than on viability. This is not surprising, because successful development requires the actions of many cellular control systems and organelles, while viability as measured by DAPI is a reflection of membrane permeability. Similar results were seen in the present study, although exposure to 40°C caused a slight reduction in the inhibitory effects of 43°C on development. Induced thermotolerance has been described for many mammalian cells [32] and is caused, at least in part, by stimulation of HSP synthesis [11-13, 33]. The beneficial effects of 40°C on subsequent responses of mouse morulae to 43°C were reduced by treatment with BSO, a specific inhibitor of y-glutamylcysteine synthetase [34]. As expected, BSO decreased embryonic GSH content of embryos exposed to either 37°C or 41°C. Thus, effects of BSO on thermotolerance are directly related to reduced concentrations of GSH, and GSH synthesis is likely required for thermotolerance. Similar findings have been reported for hamster fibroblasts [14,18] and rat postimplantation embryos [17,35]. The mechanisms by which intracellular GSH allows for induced thermotolerance have not been well defined but are probably related to its role as a free radical scavenger [36] and as an electron donor for various enzymes involved in removing reactive oxygen products and protein disulfide linkages [37]. It has not been established that heat shock increases free radical production in embryos. It is likely that it does, however, because heat shock tended to reduce GSH content in the present study; furthermore, other antioxidants, including taurine [20] and vitamin E [38], can provide partial thermoprotection to embryos. One possibility is that GSH plays a permissive role

by protecting the cell from reactive oxygen metabolites produced during heat shock and thereby allowing other biochemical changes that confer thermotolerance. It has been reported [18] that BSO decreased HSP70 synthesis in heatshocked fibroblasts although there was no effect of BSO on HSP70 mRNA or protein synthesis in postimplantation rat embryos [17]. While HSP synthesis was not monitored in the current study, there was no effect of BSO on overall protein synthesis by morulae. Treatment with SAM, an inducer of GSH synthesis [28, 29], was able to confer partial resistance to 43°C. However, SAM functions as a methyl donor in a large number of metabolic pathways [39] and could exert effects independently of effects on GSH. Also, caution is necessary in interpretation of the results of SAM treatment on the proportion of embryos that developed to the blastocyst stage. Embryos were treated with SAM for 15 h before heat shock. It might be expected, therefore, that many embryos were blastocysts before exposure to heat shock. Accordingly, some differences between control and SAM-treated embryos in response to heat shock may represent differential rates of blastocoel collapse (and subsequent classification of a collapsed blastocyst as a morula) rather than differences in the rate of development of morulae to blastocysts. Other experiments were performed to determine whether GSH or a more membrane-permeable form of GSH, monoethyl GSH ester, increased the resistance of morulae to heat shock. Both molecules were able to partially protect embryos from a heat shock of 42°C although, surprisingly, the ester, which should have had a greater effect on intracellular GSH content, was less effective than GSH itself. Other studies have also shown a thermoprotective effect of GSH for bovine [20] and mouse embryos [38]. The protection afforded by GSH and GSH ester was not complete. While these molecules increased the proportion of embryos that were classified as live based on DAPI staining, neither molecule improved development of heat-shocked embryos to a large degree. It is likely that cellular damage during heat shock involves a variety of factors besides those caused by increased free radical production and that any one thermoprotectant cannot block all effects of heat shock. Recently, free radical formation has been implicated as a detriment to the growth of non-heat-shocked embryos in culture, and administration of free radical scavengers has been reported to improve embryonic development in vitro [40-44]. In the present study, SAM, GSH, and GSH ester supplementation of medium slightly enhanced the proportion of embryos developing at 370C. While these results are consistent with the notion that production of reactive oxygen molecules could be a limiting factor to development in culture, the magnitude of the beneficial effects were small. Also, there was no deleterious effect of administration of BSO on development at 37°C even though such an effect has been reported for bovine preimplantation embryos [43]. Perhaps the effects of altering the antioxidant status of em-

ROLE OF GSH IN INDUCED THERMOTOLERANCE bryos at 37°C would have been greater if the period of culture during which antioxidant status was altered had been lengthened. In conclusion, the results support the idea that GSH is critical for the induction of thermotolerance in mouse morulae. Such a finding, as well as observations that medium supplementation with SAM and GSH reduced some of the deleterious effects of heat shock on morulae, suggests that it might be possible to limit embryonic mortality caused by maternal heat stress [1-3] by manipulating the antioxidant status of the embryo. Experiments are currently underway to test this hypothesis. REFERENCES 1. Baumgartner AP, Chrisman CL Embryonic mortality caused by maternal heat stress during mouse oocyte maturation. Anim Reprod Sci 1987; 14:309-316. 2. Wolfenson D, Blum O. Embryonic development, conception rate, ovarian function and structure in pregnant rabbits heat-stressed before or during implantation. Anim Reprod Sci 1988; 17:259-270. 3. Ealy AD, Drost M, Hansen PJ. Developmental changes in embryonic resistance to adverse effects of maternal heat stress in cows. J Dairy Sci 1993; 76:2899-2905. 4. Ulberg LC, Sheean LA. Early development of mammalian embryos in elevated ambient temperatures. J Reprod Fertil Suppl 1973; 19:155-161. 5. Gwazdauskas FC, McCaffrey C, McEvoy TG, Sreenan JM. In vitro preimplantation mouse embryo development with incubation temperatures of 37 and 39°C. J Assist Reprod Genet 1992; 9:149-154. 6. Ryan DP, Blakewood EG, Lynn JW, Munyakazi L, Godke RPAEffect of heat-stress on bovine embryo development in vitro. J Anim Sci 1992; 70:3490-3497. 7. Muller WU, Li GC, Goldstein LS. Heat does not induce synthesis of heat shock proteins or thermotolerance in the earliest stage of mouse embryo development. Int J Hyperthermia 1985; 1:97-102. 8. Ealy AD, Hansen PJ. Induced thermotolerance during early development of murine and bovine embryos. J Cell Physiol 1994; 160:463-468. 9. Li GC, Werb Z. Correlation between synthesis of heat shock proteins and development of thermotolerance in Chinese hamster fibroblasts. Proc Natl Acad Sci USA 1982; 79:3218-3222. 10. Li GC, MakJY. Re-induction of hsp70 synthesis: an assay for thermotolerance. Int J Hyperthermia 1989; 5:389-403. 11. Riabowol KT, Mizzen LA, Welch WJ. Heat shock is lethal to fibroblasts microinjected with antibodies against hsp70. Science 1988; 242:433-436. 12. HendreyJ, Kola I. Thermolability of mouse oocytes is due to the lack of expression and/or inducibility of Hsp70. Mol Reprod Dev 1991; 28:1-8. 13. Li GC, Li LG, Liu YK, Mak JY, Chen LL,Lee WM. Thermal response of rat fibroblasts stably transfected with the human 70-kDa heat shock protein-encoding gene. Proc Natl Acad Sci USA 1991; 88:1681-1685. 14. Mitchell JB, Russo A, Kinsella TJ, Glatstein E. Glutathione elevation during thermotolerance induction and thermosensitization by glutathione depletion. Cancer Res 1983; 43:987-991. 15. Konings AW, Penninga P. On the importance of the level of glutathione and the activity of the pentose phosphate pathway in heat sensitivity and thermotolerance. Int J Radiat Biol & Relat Stud Phys Chem & Med 1985; 48:409-422. 16. Skibba JL, Stadnicka A, Kalbfleish JH, Powers RH. Effects of hyperthermia on xanthine oxidase activity and glutathione levels in the perfused rat liver. J Biochem Toxicol 1989; 4:119-125. 17. Harris C, Juchau MR, Mirkes PE. Role of glutathione and Hsp 70 in the acquisition of thermotolerance in postimplantation rat embryos. Teratology 1991; 43:229239. 18. Russo A, Mitchell JB, McPherson S. The effects of glutathione depletion on thermotolerance and heat stress protein synthesis. Br J Cancer 1984; 49:753-758.

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