Induced thermotolerance in bovine two-cell embryos and the role of heat shock protein 70 in embryonic development

Induced thermotolerance is a phenomenon whereby exposure to a mild heat shock can induce heat shock proteins (HSP) and other cellular changes to make cells more resistant to a subsequent, more severe heat shock. Given that the 2-cell bovine embryo is very sensitive to heat shock, but can also produce HSP70 in response to elevated temperature, experiments were conducted to test whether 2-cell embryos could be made to undergo induced thermotolerance. Another objective was to test the role of the heat-inducible form of heat shock protein 70 (HSP70i) in development and sensitivity of bovine embryos to heat shock. To test for induced thermotolerance, 2-cell bovine embryos were first exposed to a mild heat shock (40 degrees C for 1 hr, or 41 degrees C or 42 degrees C for 80 min), allowed to recover at 38.5 degrees C and 5% (v/v) CO2 for 2 hr, and then exposed to a severe heat shock (41 degrees C for 4.5, 6, or 12 hr). Regardless of the conditions, previous exposure to mild heat shock did not reduce the deleterious effect of heat shock on development of embryos to the blastocyst stage. The role of HSP70i in embryonic development was tested in two experiments by culturing embryos with a monoclonal antibody to the inducible form of HSP70. At both 38.5 degrees C and 41 degrees C, the proportion of 2-cell embryos that developed to blastocyst was reduced (P < 0.05) by addition of anti-HSP70i to the culture medium. In contrast, sensitivity to heat shock was not generally increased by addition of antibody. In conclusion, bovine 2-cell embryos appear incapable of induced thermotolerance. Lack of capacity for induced thermotolerance could explain in part the increased sensitivity of 2-cell embryos to heat shock as compared to embryos at later stages of development. Results also implicate a role for HSP70i in normal development of bovine embryos.     

Response of preimplantation murine embryos to heat shock as modified by developmental stage and glutathione status

Summary Objectives were to characterize developmental changes in response to heat shock in the preimplantation mouse embryo and to evaluate whether ability to synthesize glutathione is important for thermal resistance in mouse embryos. Heat shock (41° C for 1 or 2 h) was most effective at disrupting development to the blastocyst stage when applied to embryos at the 2-cell stage that were delayed in development. Effects of heat shock on ability of embryos to undergo hatching were similar for 2-cell, 4-cell, and morula stage embryos. The phenomenon of induced thermotolerance, for which exposure to a mild heat shock increases resistance to a more severe heat shock, depended upon stage of development and whether embryos developed in vitro or in vivo. In particular, induced thermotolerance was observed for morulae derived from development in vivo but not for 2-cell embryos or morulae that developed in culture. Administration of buthionine sulfoximine to inhibit glutathione synthesis did not increase thermal sensitivity of 2-cell embryos or morulae but did reduce subsequent development of 2-cell embryos at both 37° and 41° C. In summary, changes in the ability of 2-cell through morula stages to continue to develop following a single heat shock were generally minimal. However, 2-cell embryos delayed in development had reduced thermal resistance, and therefore, maternal heat stress may be more likely to cause mortality of embryos that are already compromised in development. There were also developmental changes in the capacity of embryos to undergo induced thermotolerance. Glutathione synthesis was important for development of embryos but inhibition of glutathione synthesis did not make embryos more susceptible to heat shock.     

Synthesis of 60- and 72 kDa heat shock proteins in early porcine embryogenesis

Proteins of selected embryonic stages were metabolically labeled with [(35)S]-methionine and analyzed by two-dimensional SDS-polyacrylamide gel electrophoresis (2-D PAGE) to study protein expression from 4- to 8-cell to blastocyst stage of porcine embryos. Two proteins with molecular weights of 60 and 72kDa were de novo synthesized during the 4- to 8-cell stage were the earliest that were detected. They were identified as HSP60 and HSP72 according to their locations on 2-D autoradiography and the immunoblotting result of anti-HSP 60 and HSP 72 antibodies of 1-cell stage of porcine embryos. In protein translation in early pig embryogenesis the timing of their synthesis suggests that HSP60 and HSP72 play significant roles as chaperones.     

Inhibition of heat shock protein synthesis and protein glycosylation by stepdown heating

Mammalian cells exhibit increased sensitivity to hyperthermic temperatures of 38-43 degrees C after an acute high-temperature heat shock; this phenomenon is known as the stepdown heating (SDH) effect. We characterized the SDH effect on (1) the synthesis of major heat shock proteins, HSP110, 90, 72/70, 60 (35S-amino acids label), (2) on heat-induced protein glycosylation (3H-D-mannose label), and (3) on thermotolerance expression, using cell survival as an endpoint. Partitioning of label between soluble and insoluble cell fractions was separately examined. Synthesis of high molecular weight HSPs (HSP110, 90, and 72/70) was increased both by acute (10 min, 45 degrees C) and chronic (1-6 h, 41.5 degrees C) hyperthermia, primarily in the soluble cytosol fraction. SDH (10 min, 45 degrees C + 1 to 6 h, 41.5 degrees C) completely inhibited labeling of HSP110, partially inhibited HSP90 labeling, and had virtually no effect on HSP72/70 synthesis, when compared with chronic hyperthermia alone. At the cell survival level, SDH increased sevenfold the rate of cell killing at 41.5 degrees C, but reduced the expression of thermotolerance by only a factor of two. This suggests that SDH sensitization did not result from changes in HSP72/70 synthesis, nor solely from inhibition of thermotolerance. 35S-labeled HSP60 and HSP50 were found primarily in the cellular pellet fraction after both acute and chronic hyperthermia. SDH completely inhibited 35S-labeling of both HSP60 and HSP50. Labeling of GP50 with 3H-D-mannose was also completely inhibited by SDH. Moreover, SDH progressively reduced N-acetylgalactosaminyl-transferase activity. The data demonstrate that heat sensitization by SDH is accompanied by complex and selectively inhibitory patterns of HSP synthesis and protein glycosylation. Profound inhibition of HSP110, HSP60, and HSP50/GP50 labeling suggests that these may be associated with mechanisms of SDH sensitization.     

Induction of heat shock proteins in Chinese hamster ovary cells and development of thermotolerance by intermediate concentrations of puromycin

During 4 hr after puromycin (PUR: 20 micrograms/ml) treatment, the synthesis of three major heat shock protein families (HSPs: Mr = 110,000, 87,000, and 70,000) was enhanced 1.5-fold relative to that of untreated cells, as studied by one-dimensional gel electrophoresis. The increase of unique HSPs, if studied with two-dimensional gels, would probably be much greater. In parallel, thermotolerance was observed at 10(-3) isosurvival as a thermotolerance ratio (TTR) of either 2 or greater than 5 after heating at either 45.5 degrees C or 43 degrees C, respectively. However, thermotolerance was induced by only intermediate concentrations (3-30 micrograms/ml) of puromycin that inhibited protein synthesis by 15-80%; a high concentration of PUR (100 micrograms/ml) that inhibited protein synthesis by 95% did not induce either HSPs or thermotolerance. Also, thermotolerance was never induced by any concentration (0.01-10 micrograms/ml) of cycloheximide that inhibited protein synthesis by 5-94%. Furthermore, after PUR (20 micrograms/ml) treatment, the addition of cycloheximide (CHM: 10 micrograms/ml), at a concentration that reduces protein synthesis by 94%, inhibited both thermotolerance and synthesis of HSP families. Thus, thermotolerance induced by intermediate concentrations of PUR correlated with an increase in newly synthesized HSP families. This thermotolerance phenomenon was compared with another phenomenon termed heat resistance and observed when cells were heated at 43 degrees C in the presence of CHM or PUR immediately after a 2-hr pretreatment with CHM or PUR. Heat protection increased with inhibition of synthesis of both total protein and HSP families. Moreover, this heat protection decayed rapidly as the interval between pretreatment and heating increased to 1-2 hr, and did not have any obvious relationship to the synthesis of HSP families. Therefore, there are two distinctly different pathways for developing thermal resistance. The first is thermotolerance after intermediate concentrations of PUR treatment, and it requires incubation after treatment and apparently the synthesis of HSP families. The second is resistance to heat after CHM or PUR treatment immediately before and during heating at 43 degrees C, and it apparently does not require synthesis of HSP families. This second pathway not requiring the synthesis of HSP families also was observed by the increase in thermotolerance at 45.5 degrees C caused by heating at 43 degrees C after cells were incubated for 2-4 hr following pretreatment with an intermediate concentration of PUR.     

Inhibition of heat shock protein synthesis and thermotolerance by cycloheximide

Chinese hamster ovary (CHO) cells were exposed to a 43 degrees C, 15-min heat shock to study the relationship between protein synthesis and the development of thermotolerance. The 43 degrees C heat shock triggered the synthesis of three protein families having molecular weights of 110,000, 90,000, and 65,000 (HSP). These proteins were synthesized at 37 and 46 degrees C. This heat shock also induced the development of thermotolerance, which was measured by incubating the cells at 46 degrees C 4 h after the 43 degrees C heat treatment. CHO cells were also exposed to 20 micrograms/ml of cycloheximide for 30 min at 37 degrees C, 15 min at 43 degrees C, and 4 h at 37 degrees C. This treatment inhibited the enhanced synthesis of the Mr 110,000, 90,000, and 65,000 proteins. The cycloheximide was then washed out and the cells were incubated at 46 degrees C. HSP synthesis did not recover during the 46 degrees C incubation. This cycloheximide treatment also partially inhibited the development of thermotolerance. These results suggest that for CHO cells to express thermotolerance when exposed to the supralethal temperature of 46 degrees C protein synthesis is necessary.     

Differential responses of bovine oocytes and preimplantation embryos to heat shock

The authors sought to determine whether developmental differences in the magnitude of embryonic mortality caused by heat stress in vivo are caused by changes in resistance of embryos to elevated temperature. In this regard, responses of oocytes, two-cell embryos, four- to eight-cell embryos, and compacted morulae to heat shock were compared. An additional goal was to define further the role of cumulus cells and glutathione in thermoprotection of oocytes. In experiment 1, heat shock (41°C for 12 hr) decreased the number of embryos developing to the blastocyst stage for two-cell (26% vs. 0%) and four- to eight-cell (25% vs. 10%) embryos but did not affect morulae (37% vs. 42%). In experiment 2, exposure of two-cell embryos to 41°C for 12 hr reduced the number of four- to eight-cell embryos present 24 hr after the end of heat shock (88% vs. 62%). In experiment 3, heat shock reduced the number of two-cell embryos developing to blastocyst (49% vs. 8%) but did not affect subsequent development of oocytes when heat shock occurred during the first 12 hr of maturation (46% vs. 41% development to blastocyst); membrane integrity was not altered. In experiment 4, oocytes were cultured with an inhibitor of glutathione synthesis, _DL-buthionine-[S,R]-sulfoximine (BSO), for 24 hr and exposed to 41°C for the first 12 hr of maturation. Percentages of blastocysts were 35% (39°C), 18% (41°C), 17% (39°C+BSO), and 11% (41°C+BSO). For experiment 5, oocytes were either denuded or left with cumulus intact and were then radiolabeled with [³⁵S]methionine and [³⁵S]cysteine at 39°C or 41°C for 12 hr. Exposure of oocytes to 41°C for 12 hr reduced overall synthesis of ³⁵S-labeled TCA-precipitable intracellular proteins (18,160 vs. 14,594 dpm/oocyte), whereas presence of cumulus increased synthesis (9,509 vs. 23,246). Analysis by two-dimensional SDS PAGE and fluorography revealed that heat shock protein 68 (HSP68) and two other putative heat shock proteins, P71 and P70, were synthesized by all oocytes regardless of treatment. Heat shock did not alter the synthesis of HSP68 or P71 but decreased amounts of newly synthesized P70. Cumulus cells increased synthesis of P71 and P70. Results indicate there is a biphasic change in resistance to elevations in temperature as oocytes mature, become fertilized, and develop. Resistance declines from the oocyte to the two-cell stage and then increases. Evidence suggests a role for cumulus cells in increasing HSP70 molecules and protein synthesis. Data also indicate a role for glutathione in oocyte function. Mol Reprod Dev 46:138–145, 1997. © 1997 Wiley-Liss, Inc.     

Induction of the heat shock response and translational thermotolerance in day 15 ovine trophectoderm

The objectives of this study were to determine the ability of trophectoderm from preimplantation ovine embryos to synthesize hsp70 in response to heat shock and to identify conditions which induce translational thermotolerance in this tissue. Day 15 embryos were collected, and proteins synthesized in 1.5-mm sections of trophectoderm were radioactively labeled with (35)S-methionine. One-dimensional SDS-PAGE gels, two-dimensional gel electrophoresis and Western blots were utilized to characterize the heat shock response and to examine the induction of translational thermotolerance. Increased synthesis of the 70 kDa heat shock proteins and a protein with an approximate molecular weight of 15 to 20 kDa was observed with heat shock (> or = 42 degrees C). Total protein synthesis decreased (P < 0.05) with increased intensity of heat shock. At 45 degrees C, protein synthesis was suppressed with little or no synthesis of all proteins including hsp70. Recovery of protein synthesis following a severe heat shock (45 degrees C for 20 min) occurred faster (P < 0.05) in trophectoderm pretreated with a mild heat shock (42 degrees C for 30 min) than trophectoderm not pretreated with mild heat. In summary, trophoblastic tissue obtained from ovine embryos exhibit the characteristic "heatshock" response similar to that described for other mammalian systems. In addition, a sublethal heat shock induced the ability of the tissue to resume protein synthesis following severe heat stress. Since maintaining protein synthesis is crucial to embryonic survival, manipulation of the heat-shock response may provide a method to enhance embryonic survival.     

Influence of temperature on the development and decay of thermotolerance and heat shock proteins

The development of thermotolerance and its decay in plateau-phase Chinese hamster cells are shown to be temperature-dependent phenomena. Development of tolerance, after an initial dose of 10 min at 45 degrees C, is appreciably slower between 20 and 28 degrees C than it is at 37 degrees C. Decay of tolerance is also slower in that temperature range; at 4-23 degrees C, it does not decay at all during the 96-h interval of the experiment. At 41 degrees C, thermotolerance decay, "step-down" cell killing, and thermotolerance induction apparently all occur and affect cell survival. The decay of HSP 70 mirrors that of thermotolerance, except at 41 degrees C. At that temperature very likely de novo synthesis of that protein becomes important in determining protein concentration. Our data show that care must be taken when extrapolating from kinetic data obtained with surface tissues in vivo to those in depth. The former are usually at a temperature between 25 and 32 degrees C; the latter are at 37 degrees C.     

Developmental regulation of heat shock protein synthesis and HSP 70 RNA accumulation during postimplantation rat embryogenesis

Exposure of postimplantation rat embryos on days 9, 10, 11, and 12 of gestation to an in vitro heat shock of 43 degrees C for 30 min results in the induction of heat shock proteins (HSPs) in day 9 and 10 embryos, a severely attenuated response in day 11 embryos, and no detectable response in day 12 embryos. The heat shock response in day 9 embryos (presomite stage) is characterized by the synthesis of HSPs with molecular weights of 28-78 kDa. In heat shocked day 10 embryos, two additional HSPs are induced (34 and 82 kDa). In addition, two HSPs present on day 9 are absent on day 10. In day 11 heat shocked embryos, only three HSPs (31, 39, and 69 kDa) are induced, while in day 12 embryos no detectable HSPs are induced. Northern blot analysis of HSP 70 RNA levels indicates that the accumulation of this RNA, but not actin RNA, varies depending on developmental stage at the time of exposure to heat as well as the duration of the heat shock. Day 9 embryos exhibit the most pronounced accumulation of HSP 70 RNA while embryos on days 10-12 exhibit an increasingly attenuated accumulation of HSP 70 RNA, particularly after the more acute exposures (43 degrees C for 30 or 60 min). Thus, the ability to synthesize HSP 70 and to accumulate HSP 70 RNA changes dramatically as rat embryos develop from day 9 to day 12 (presomite to 31-35 somite stages).     

Nuclear-mitochondrial cross-talk during heat shock in Arabidopsis cell culture

Apart from energy generation, mitochondria perform a signalling function determining the life and death of a cell under stress exposure. In the present study we have explored patterns of heat-induced synthesis of Hsp101, Hsp70, Hsp17.6 (class I), Hsp17.6 (class II) and Hsp60, and the development of induced thermotolerance in Arabidopsis thaliana cell culture under conditions of mitochondrial dysfunction. It was shown that treatment by mitochondrial inhibitors and uncouplers at the time of mild heat shock downregulates HSP synthesis, which is important for induced thermotolerance in plants. The exposure to elevated temperature induced an increase in cell oxygen consumption and hyperpolarization of the inner mitochondrial membrane. Taken together, these facts suggest that mitochondrial functions are essential for heat-induced HSP synthesis and development of induced thermotolerance in A. thaliana cell culture, suggesting that mitochondrial-nuclear cross-talk is activated under stress conditions. Treatment of Arabidopsis cell culture at 50 degrees C initiates a programmed cell death determined by the time course of viability decrease, DNA fragmentation and cytochrome c release from mitochondria. As treatment at 37 degrees C protected Arabidopsis cells from heat-induced cell death, it may be suggested that Hsp101, Hsp70 and small heat-shock proteins, the synthesis of which is induced under these conditions, are playing an anti-apoptotic role in the plant cell. On the other hand, drastic heat shock upregulated mitochondrial Hsp60 synthesis and induced its release from mitochondria to the cytosol, indicating a pro-apoptotic role of plant Hsp60.     

Hyperthermia-induced heat shock response and thermotolerance in postimplantation rat embryos

Postimplantation stage rat embryos (6-10 somites) undergo abnormal development after exposure to a temperature of 43 degrees C for 30 min. A heat shock of 43 degrees C for 30 min also induces the synthesis of a set of eight heat shock proteins (hsps) with molecular masses ranging from 28,000 to 82,000 Da. The synthesis of these hsps is rapidly induced after the heat shock is applied and rapidly decays after embryos are returned to 37 degrees C. A heat shock of 42 degrees C for 30 min has no effect on rat embryo growth and development, but does induce the synthesis of three hsps. The most prominent of these three is believed to be the typical mammalian 70 kDa hsp. Furthermore, a 42 degrees C, 30-min heat shock followed by a 43 degrees C 30-min heat shock leads to partial protection from the embryotoxic effects of a single exposure at 43 degrees C, i.e., thermotolerance.     

Heat-Induced Changes in Thermosensitivity and Gene Expression during Development

The thermosensitivity of developing embryos of the fresh water snail Lymnaea stagnalis was investigated from the 4-cell stage to the 3-day-old trochophore larva by means of survival curves for 43.6°C. Cleavage stage embryos were extremely thermoresistant as compared with older stages, and thermosensitivity increases during the development.
Pretreatment with a mild heat exposure (10 min at 39°C) did not induce thermotolerance at the 4-cell stage, but it did so in the early gastrula and trochophora. Development of thermotolerance in 1-, 2-, and 3-day-old stages showed an identical kinetic pattern.
After incubation in ³⁵S-methionine one-dimensional gel electrophoresis was carried out with or without preheating. At the 4-cell stage no enhanced synthesis of heat shock proteins was induced by exposure to heat. At stages of 1 day and older heat induced the enhanced synthesis of the heat shock proteins with apparent molecular weights of 38, 65 and 70 kilodaltons. The synthesis of heat shock protein 70 changes during the early development of Lymnaea both in its constitutive level and in its ability to be enhanced by heat treatment.     

Responsiveness of early embryos to environmental insults: potential protective roles of HSP70 and glutathione

Early embryos are not as passive as previously thought. In the bovine, embryos as early as the 2-cell stage can respond to environmental insults at both the cellular and molecular level by altering expression of specific genes and synthesis of proteins. Moreover, sex related differences exist in how early embryos respond to otherwise hostile environments. As aggressive as early embryos may be to tolerate environment insults, the majority will fail to continue in development. Reduced developmental potential of embryos exposed to elevated temperatures is likely due to direct effects on the early embryo. However, as embryos proceed in development they acquire the ability to better withstand environmental insults. Developmental acquisition of tolerance to environmental stress may be contingent upon acquisition of protective biochemical mechanisms or simply due to increased cell numbers. Correlative evidence has suggested a potential role of heat shock protein 70 and glutathione for protection of embryos in face of elevated temperature. Of these two possibilities, HSP70 appears least likely to play a significant role in developmental acquisition of thermotolerance. Bovine embryos as early as the 2-cell stage, are able to mount a heat shock response; a developmental stage that is most sensitive to elevated temperatures. A more likely candidate for conferring increased resistance of early embryos to elevated temperature is glutathione.     

Induced thermotolerance during early development of murine and bovine embryos

During early development, elevated temperatures have deleterious effects on embryonic viability and development. The primary objective of the current study was to determine the ontogeny of induced thermotolerance during early murine embryonic development. Embryos were either retrieved from superovulated ICR female mice at the 2 cell and 4 cell stages and cultured thereafter or were retrieved from oviducts or uterine horns at the desired stage of development. Induction of thermotolerance was detected by evaluating viability and further development after embryos were exposed to homeothermic temperature (37°C), mild heat shock (40°C for 1 h), severe heat shock (42°C for 1 h or 43°C for 2 h), or mild heat shock followed by severe heat shock (to induce thermotolerance). Induction of thermotolerance was observed beginning at the 8 cell stage when embryos were developed in culture from the 2 cell to 4 cell stage. When embryos were developed in vivo (i.e., were retrieved from the reproductive tract at the desired stage of development), thermotolerance was not induced until the blastocyst stage of development. The induction of thermotolerance was dependent on serum supplementation since induction of thermotolerance was not observed when embryos were placed in medium without serum. Induced thermotolerance could also be demonstrated in bovine blastocysts. In conclusion, embryos acquire the ability to undergo thermotolerance as they progress through development. The timing of processes leading to acquisition of thermotolerance can, however, be hastened by exposure of embryos to in vitro conditions.     

Developmental changes in inhibitory effects of arsenic and heat shock on growth of pre-implantation bovine embryos

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 > or = 16-cells in number) to either sodium arsenite (0, 1, 5, or 10 microM) or heat shock (exposure to 41 degrees C for 0, 3, 4.5, 6, or 9 hr). Arsenic induced apoptosis and increased group 2 caspase activity for embryos at the > or = 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 > or = 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 > or = 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.