Evidence That Glutathione is Involved in Thermotolerance Of Preimplantation Murine Embryos

BIOLOGY OF REPRODUCTION 52, 1296-1301 (1995) Evidence That Glutathione Is Involved in Thermotolerance of Preimplantation Murine Embryos' C.F. ARECHIGA, A.D. EALY, 3 and PJ. HANSEN 2 Departmentof Dairy and Poultry Sciences, University of Florida, Gainesville, Florida 32611-0920 ABSTRACT 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 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. ROLE OF GSH IN INDUCED THERMOTOLERANCE +/- BSO 37 C i: :: ::::i::: i :d :: : :: d __:: dfi7 _i :E _ 37 C I 40 C 40 C 43 C 43 C 40/43 40 C I 1 I I I 2 3 4 43 C 5 I I I I 6 7 8 9 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) cont (...truncated)