EFFICACY OF VITAMIN E AND GLUTATHIONE FOR THERMOPROTECTION OF MURINE MORULAE. C. F. ArBchiga, A. D. Ealy* and P. J. Hansen3

Transcription

1 Theriogenology41: ,1994 EFFICACY OF VITAMIN E AND GLUTATHIONE FOR THERMOPROTECTION OF MURINE MORULAE C. F. ArBchiga, A. D. Ealy* and P. J. Hansen3 Department of Dairy and Poultry Sciences Animal Molecular and Cell Biology Program University of Florida Gainesville, FL , USA Received for publication: October 20, 1993 Accepted: March 8, 1994 ABSTRACT The objective of this study was to determine whether vitamin E and glutathione could limit deleterious effects of elevated temperature on viability and development of murine morulae. Exposure of morula-stage embryos to heat shock (42 C for 1 h or 43 C for 2 h) decreased the proportion of live embryos at the end of culture and the proportion that developed to the blastocyst stage. In two experiments, administration of vitamin E reduced adverse effects of heat shock on viability as determined by dye exclusion. Vitamin E did not, however, block the inhibitory effect of heat shock on the proportion of embryos that developed to the blastocyst stage. In 2 other experiments, glutathione reduced the effects of heat shock on viability but did not have any consistent beneficial effect on the proportion of heat-shocked embryos that became blastocysts. Vitamin E and glutathione can partially protect embryos from detrimental effects of heat shock. This protective effect is not sufficient, however, to allow for continued development under the conditions utilized. Key words: Vitamin E, glutathione, embryonic development, thermoprotection INTRODUCTION Viability and development of preimplantation mammalian embryos is compromised by exposure to elevated temperature (4,5,7,12,20,21,26). Detrimental effects of heat shock are probably related, at least partially, to an increased generation of free radicals and peroxides in Acknowledgments This paper is Journal Series No. R of the Florida Agricultural Experiment Station. Research supported by USDA CBAG Grant No and a grant from the Florida Dairy Checkoff Program. The authors thank Mrs. M. E. Hissem for preparation of the manuscript. Supported by the CONACYT/IIE Grants Program (Mexico) and CIBMYC (International Center for Cell and Molecular Biology, A.C.), Monterrey, N.L., Mexico. *Present address: Department of Animal Sciences, University of Missouri, Columbia, MO USA. 3Correspondence and reprint requests. Copyright Butterworth-Heinemann

2 1546 Theriogenology response to elevated temperatures (11). Inhibition of synthesis of one antioxidant, glutathione, makes preimplantation-stage mouse embryos unable to undergo induced thermotolerance (3). Given the potential importance of free radicals in causing cellular damage during heat shock, it may be feasible to limit effects of elevated tem~~tures on embryonic survival of heat-stressed females by exogenous administ~tion of antioxidants. In support of this idea are results that the administration of glutathione reduced the effects of heat shock on cultured bovine morulae (4). The objective of the present study was to use cultured mouse embryos as a model to determine whether 2 antioxidants, vitamin E and glutathione, can provide thermoprotection to heat-shocked embryos. Vitamin E maintains the integrity of membrane phospholipids against oxidative damage and peroxidation (13,15). Glutathione (GSH), an antioxidant present in mouse preimpi~~tion embryos at millimolar concentrations (16), acts by directly reacting with free radicals and by p~icipating in various enzymatic reactions that eliminate free radicals and protein disulfide bonds (8,14). Materials MATERIALS AND METHODS Silicon oil was purchased from Aldrich Chemical Co. (Miiwaukee, WI, USA). Reduced glu~thione, (+)-~-t~opherol acid succinate (vitamin E), succinic acid, 4,6 -diamidino-2- phenylindole (DAPI) and heat-treated fetal calf serum (htfcs) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Superovulation, Embryo Collection and Culture Mice of the ICR strain (Harlan Sprague Dawley Inc., Indianapolis, IN, USA) were superovulated as previously described (12). Embryos were collected at the 2-cell stage and cultured in groups of 5 to 10 in 5 to 7-~1 microdrops of Ml % BSA covered with twiceextracted silicon oil. The emb~os were cultured at 37 C with an atmosphere of 5% COz in humid air until they reached the morula stage. Morulae were then transferred in groups of 5 to 10 into 5~1 microdrops of Ml6 medium + 10% htfcs and other additives as described for each experiment. Effect of Vitamin E on Responses of Morulae to Heat Shock For Experiment 1, morulae were cultured in Ml6 medium -t 10% htfcs (i.e., negative control); 250 FM succinic acid (i.e., vehicle control); 25 (*M vitamin E [(+)-~-t~oph~rol acid succinate] + succinic acid; or 250 PM vitamin E. After 4 h of vitamin E treatment, embryos were exposed to either 37 C continuously for 20 h or to a heat-shock of 43 C for 2 h followed by 37 C for 18 h. The embryos were then assessed for viability and stage of development. For Experiment 2, morula-stage embryos were placed at 37 C in Ml6 medium containing 10% htfcs PM vitamin E. After 4 h of culture, embryos were either cultured at 37 C continuously for 20 h, exposed to a heat shock of 42 C for 1 h, or exposed to a severe heat shock of 43 C for 2 h. Heat-shocked embryos were then incubated at 37 C for 19 and 18 h, respectively and then assessed for viability and development.

3 Theriogenology 1547 Effect of GSH on Responses of Morulae to Heat Shock For Experiment 3, morulae were placed in 5- to lo-p1 microdrops of Ml6 medium + 10% htfcs + 5 PM GSH. After 2 h at 37 C, embryos were incubated at either 37 C or 43 C for 2 h. Embryos were then cultured for an additional 20 h at 37 C before evaluation for viability and development to blastocyst. Experiment 4 was performed similarly except that concentrations of GSH were 0, 50 nm or 5 FM. Determination of Viability and Development Viability (i.e., the proportion of cells that are live based on dye exclusion) was determined by use of the fluorescent compound, DAPI (23). Embryos were placed in Dulbecco s PBS containing % (w/v) DAPI. After 15 to 20 min, embryos were evaluated for fluorescing nuclei using an epifluorescent microscope with ultraviolet excitation and a 490 nm emission filter. Embryos were considered alive if less than approximately one-third of the cells fluoresced and as dead if more than one third fluoresced. Embryonic development was determined by visual observation during DAPI staining. Embryos were classified as having progressed in development if they advanced from the morula stage to the blastocyst stage. Statistical Analysis Data were analyzed by least-squares analysis of variance using the General Linear Models procedure of SAS (22). Least squares analysis of variance procedures were utilized as suggested by Wilcox et al. (27). Experiments were performed on several occasions. For initial analyses, effects of day and interaction with day were included in the model. Those effects were subsequently removed if they did not improve the analysis. For Experiment 1, data were analyzed as a 2 x 4 factorial design, including main effects of temperature (37oC vs. 43 C) and the 4 treatments. Orthogonal contrasts (control + succinic acid vs FM vitamin E, control vs succinic acid, 25 vs 250 PM vitamin E) were used to resolve effects of treatment and temperature-by-treatment into individual comparisons. For Experiment 2, data were analyzed as a 2 x 3 factorial design, considering the main effect of vitamin E and temperature (37 C 42 C and 43 C). For Experiment 3, data were analyzed as a 2 x 2 factorial design with main effects of temperature (37 C vs. 43 C) and glutathione. For Experiment 4, data were analyzed using a 3 x 3 factorial design with main effects of temperature (37 C 42 C/l h, 43 C/2 h) and dose of glutathione (0, 50 nm, 5 PM). Effects of Vitamin E RESULTS In Experiment 1, exposure to 43 C reduced (P < 0.001) viability and percentage of embryos that developed to the blastocyst stage. There was a significant temperature x vitamin E interaction affecting viability (P<O.Ol; Table 1). This occurred because presence of vitamin E reduced the deleterious effects of heat shock on viability. In the absence of vitamin E, viability was decreased from 100% at 37 C to 40% (negative control) or 50% (250 PM succinic acid) for embryos exposed to 43 C. However, for embryos treated with 25 and 250 PM vitamin E, 69 and 78% of embryos, respectively, were alive after exposure to 43 C. The

4 1548 Theriogenology thermoprotective effect of vitamin E did not allow for increased development after 43 C. In fact, vitamin E tended to reduce the percentage of embryos that reached blastocyst stage at 43 C but not at 37 C (temperature-by-control + succinic acid vs PM vitamin E interaction; P<O.O5). In Experiment 2, exposure of embryos to both heat shocks reduced viability of embryos at the end of culture (P<O.OOl) and percentage of embryos that became blastocysts (P<O.OOl; Table 2). As expected, exposure of embryos to 43 C for 2 h caused a greater decrease than 42 C for 1 h. There was a temperature-by-vitamin E interaction for viability (P<O.OOl) because the deleterious effects of both heat shocks were reduced in embryos treated with vitamin E. Vitamin E did not abrogate effects of 42 C or 43 C on the proportion of embryos that developed to the blastocyst stage. Table 1. Effect of vitamin E on viability (based on dye exclusion) and development of murine morulae exposed to a heat shock of 43 C for 2 hours Temperature Treatment No. of % live at 24 % blastocysts embryos hours? at 24 hoursb 37 C Control C 250 /.&I Succinic Acid C 25 PM Vitamin E C 250 /LM Vitamin E C 43 C 43 C 43 C Control 250 /AM Succinic Acid 25 PM Vitamin E 250 /LM Vitamin E Significant effects: temperature (P < O.OOl), treatment (P < 0.05) and temperature-by-treatment (P<O.Ol). Using orthogonal contrasts, control + succinic acid differed from 25,uM mm vitamin E (P<O.Ol). The temperature-by-control + succinic acid vs PM vitamin E interaction was significant (P<O.OOl). Significant effects: temperature (P < 0.001). Using orthogonal contrasts, there was a temperature-by-control + succinic acid vs PM vitamin E interaction (P<O.O5).

5 Theriogenology 1549 Table 2. Effect of vitamin E on viability (based on dye exclusion) and subsequent development of murine morulae exposed to heat shock of 42 C for 1 hour or 43 C for 2 hours Temperature Vitamin E (PM) No. of embryos % live at 24 hoursa % blastocyst at 24 hoursb 37 C C C/l h Wl h W2 h W2 h a Significant effects: temperature, vitamin E and temperature-by-vitamin E (P < 0.001). b Significant effects: temperature (P < ). Table 3. Effect of 5 PM GSH on viability (based on dye exclusion) and development of murine morulae exposed to a heat shock of 43 C for 2 hours Temperature Treatment No. of % alive at % blastocyst embryos 24 hoursa at 24 hoursb 37 C Control C GSH C Control oc GSH asignificant effects: temperature, treatment and temperature-by-treatment (P < 0.001). bsignificant effects: temperature (P < 0.01) and temperature-by-treatment (P < 0.05). Effects of GSH In Experiment 3, exposure to 43 C reduced viability (P<O.OOl) and the percentage of development to the blastocyst stage (P <O.Ol; Table 3). Glutathione reduced the effect of 43 C on viability (temperature-by-treatment interaction P < 0.001). There was also a temperature-bytreatment interaction (P < 0.05) for development to the blastocyst stage. Glutathione caused a

6 1550 Theriogenology slight decrease in the proportion of embryos becoming blastocysts at 37 C (64 vs 58%; control vs GSH) whiie causing a slight increase at 43 C (10 vs 15%). For Experiment 4 (Table 4), GSH reduced the effect of 43 C on viability in a dose-dependent manner ~tem~rature-by-tr~tment inte~ction; P<O.OS) but did not improve development at 43 C. Rather, GSH improved development for embryos at 37 C only (tem~rature-by-~~tment interaction; P < 0.001). Table 4. Dose-dependent effect of glutathione on viability (based on dye exclusion) and develodment of murine morulae exuosed to a heat shock of 43 C for 2 hours Temperature GSH No. of embryos % live at 24 hours % developed at 24 hoursb 37 C C 50 nm C 5 CLM C C 50 nm C 5 PM asignificant effects: temperature (P < O.OOl), treatment (P < 0.05) and temperature-by-treatment (PCO.05). bsignificant effects: temperature, treatment and temperature-by-treatment (P<O.OOl). DISCUSSION These experiments were performed to determine whether provision of molecules with antioxidant properties would protect embryos from heat shock. It was reasoned that these molecules would reduce oxidative damage caused by free radicals and peroxides generated during heat shock (11) and would therefore minimize deleterious effects of heat shock on embryonic development. The two antioxidants tested were vitamin E, which functions as the chief antioxidant in membrane compartments of the cell (13,15) and GSH, which is an important cytoplasmic ~tioxid~t (8,14) present in millimolar concentrations in p~impl~~tion embryos (16). Results indicate that both molecules were able to reduce the deleterious effects of heat shock on embryonic function but neither molecule was able to completely overcome effects of heat shock. In the present study, both vitamin E and GSH reduced deleterious effects of heat shock on viability while generally having no beneficial effect on development of heat-shocked blastocysts. Other studies with mouse preimplantation embryos indicate that developmental processes are more disrupted by heat shock than viability and that effects of heat shock on development are less amenable to thermoprot~tion than effects on viability. Eaty et al. (5) found that exposure

7 of preimplantation embryos to a heat shock of 42 C for 1 h caused no effect on viability but blocked subsequent development. Moreover, thermotolerance in preimplantation stage mouse embryos induced by exposure to a mild heat shock (4OT for 1 h) caused greater resistance to a sub~uent, more severe heat shock with respect to viabili~ than to development potential (3,5). It is not surprising that development is more perturbable by heat shock and therefore less amenable to thermoprotective protocols. While viability as determined in the current studies requires only that embryonic cells have intact plasma membranes, proper development requires several cellular and molecular processes to take place in a coordinated fashion. In contrast to the present results, Ealy et al. (4) found that GSH blocked effects of exposure to 42 C on viability and development of bovine morula. It is possible that bovine embryos differ from mouse embryos in response to heat shock or antioxidant treatment. Additionally, the thermoprotective effect of GSH in the present study was evaluated for embryos exposed to 43 C rather than 42 C as evaluated by Ealy et al. (4). Free radical formation has been implicated as a detriment to development of preimpl~~tion embryos even in the absence of heat shock, and it has been suggested that such molecules might be responsible for the 2-cell block to development in cultured mouse embryos (6,9,17,18). Provision of various molecules involved in removal of free radicals has been reported to increase development of mouse (6,9,18), rabbit (10) and bovine (24) embryos. In contrast, there was generally no beneficial effect of vitamin E or GSH on non heat-stressed embryos in the present study. The exception was for Experiment 4, where GSH caused an increase in the proportion of embryos at 37 C that became blastocysts. In other studies (6,9,1~,18,24), ~tioxid~ts were provided at earlier stages of devdopment than in the present study. This suggests that use of antioxidants to promote embryonic development is most effective when molecules are provided early in development. The long-term objective of this research is to develop methods for using antioxidants to improve emb~onic survival in heat-stressed females. Early pregn~~y loss due to heat stress can be severe in domestic animals (25). Since hy~~he~ia in vivo differs from the heat shock used to treat embryos in culture (i.e., is more gradual, usually less severe, and usually of a greater duration), it is possible that the effectiveness of antioxidants in vivo may be different than for cultured embryos. Nonetheless, the fact that the thermoprotection afforded by vitamin E and GSH was not complete indicates that use of these antioxidants to improve embryonic survival in heat-stressed females is unlikely to be successful unless the approach is modified from that taken here. Perhaps, a more effective scheme would be to administer a mixture of antioxidants to exert antioxidant effects at several extracellular and intracellular compartments. In the present study, GSH probably acted at the extracellular level, since intracellular concentrations of GSH in mouse embryos (0.5 to 10 mm; 16) are too high to be affected by concentrations in medium. Moreover, GSH is not readily transported across cells (14). Therefore, membrane-soluble analogs of GSH might be more effective as thermoprotectants. Monoesters of GSH, which are more memb~ne-soluble than GSH (2,19)% have been shown to be more effective than GSH in blocking pathological effects caused by the drug, cisplatin (I). Even though thermoprotection was not complete in the present study, results are encouraging because they demonstrate that antioxidant supplementation can reduce some effects of heat

8 1552 Theriogenology shock. Further research should be directed towards an optimization of antioxidant therapy using cultured embryos followed by field trials to determine whether provision of antioxidants has beneficial effects on the fertility of females that become hyperthermic early in pregnancy. REFERENCES 1. Anderson ME, Naganuma A, Meister A. Protection against cisplatin toxicity by administration of glutathione ester. FASEB J 1990;4: Anderson ME, Powrie F, Puri RN, Meister A. Glutathione monoethyl ester: preparation, uptake by tissues, and conversion to glutathione. Arch Biochem Biophys : Ar&higa CF, Ealy AD, Hansen PJ. Induction of thermotolerance in mouse embryos is blocked by the glutathione synthesis inhibitor, DL-buthionine-[S,R]-sulfoximine(BS0). J Anim Sci (Suppl 1):265 (abstr). 4. Ealy AD, Drost M, Barros CM, Hansen Pi. Thermoprotection of preimplantation bovine embryos from heat shock by glutathione and taurine. Cell Biol Intl Rep 1992; 16: Ealy AD, Hansen PJ. Induced thermotolerance during early development of murine and bovine embryos. J Cell Physiol (accepted). 6. Goto Y, Noda Y, Narimoto K, Umaoka Y, Mori T. Oxidative stress on mouse embryo development in vitro. Free Rad Biol Med 1992;13: Gwasdauskas 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: Kosower EM. Chemical properties of glutathione. In: Arias IM, Jakoby WI3 (eds), Glutathione: Metabolism and Function. New York, Raven Press, 1976; Legge M, Sellens MH. Free radical scavengers ameliorate the 2-cell block in mouse embryo culture. Human Reprod 1991;6: Li J, Foote RI-I, Simkin M. Development of rabbit zygotes cultured in protein-free medium with catalase, taurine, or superoxide dismutase. Biol Reprod 1993;48: Loven DP. A role for reduced oxygen species in heat induced cell killing and the induction of thermotolerance. Med Hypotheses 1988;26: Malayer JR, Pollard JW, Hansen PJ. Modulation of thermal killing of bovine lymphocytes and preimplantation mouse embryos by alanine and taurine. Am J Vet Res 1992;53: McCay PB, King MM. Vitamin E: its role as a biologic free radical scavenger and its relationship to the microsomal mixed-function oxidase system. In: Machlin IJ (ed), Vitamin E: A Comprehensive Treatise. New York, Marcel Deklcer Inc, 1980; Meister A. Selective modification of glutathione metabolism. Science 1985;220: Molenaar I, Hulstaret CE, Hardonk MJ. Role in function and ultrastructure of cellular membranes. In: Machlin LJ (ed), Vitamin E: A Comprehensive Treatise. New York, Marcel Dekker Inc, 1980; Nasr-Esfahani MI-I, Johnson MH. Quantitative analysis of cellular glutathione in early preimplantation mouse embryos developing in vivo and in vitro. Human Reprod 1992;7: Nasr-Esfahani MH, Winston NJ, Johnson MH. Effects of glucose, glutamine, ethylenediamine tetraacetic acid and oxygen tension on the concentration of reactive oxygen species and on development of the mouse preimplantation embryo in vitro. J Reprod Fertil 1992;96: Nasr-Esfahani MH, Johnson MH. How does transferrin overcome the in vitro block to development of the mouse preimplantation embryo? J Reprod Fertil 1992;96:41-48.

9 Theriogenology 19. Puri RN, Meister A. Transport of glutathione, as y-glutamylcysteinylglycyl ester, into liver and kidney. Proc Natl Acad Sci USA 1983;80: Putney DJ, Drost M, Thatcher WW. Embryonic development in superovulated dairy cattle exposed to elevated ambient temperatures between days 1 to 7 postinsemination. Theriogenology 1988;30: Ryan DP, Blakewood EG, Lynn JW, Munyakazi L, Godke RA. Effect of heat-stress on bovine embryo development in vitro. J Anim Sci 1992;70: SAS. Statistical Analysis System: A User s Guide. SAS Inst, Version 6, 4th edition, Cary NC, Schilling VE, Niemann H, Cheng SP, Doepke HH. DAPI - A further fluorescence test for diagnosing the viability of early cow and rabbit embryos. Ziichthygiene 1979; 14: Takahashi M, Nagai T, Hamano S, Kuwayama M, Okamura N, Okano A. Effect of thiol compounds on in vitro development and intracellular glutathione content of bovine embryos. Biol Reprod 1993;49: Thatcher WW, Hansen PJ. Environment and reproduction. In: King GJ (ed) Reproduction in Domesticated Animals. Elsevier, 1993; Ulberg LC, Sheean LA. Early development of mammalian embryos in elevated ambient temperatures. J Reprod Fertil 1973;19(Suppl):l Wilcox CW, Thatcher WW, Martin FG. Statistical analysis of repeated measurements in physiology experiments. In: Livestock Reproduction in Latin America, Int Atomic Energy Agency, Vienna, 1990;