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.     

Regulation of heat shock protein 70 synthesis by heat shock in the preimplantation murine embryo

Induced thermotolerance in murine embryos occurs at the 8-cell stage when embryos are maintained in vitro but not until the blastocyst stage if development proceeds in vivo. Present results indicate that ability of embryos to undergo induced thermotolerance is not limited by heat shock protein 70 (HSP70) synthesis. Exposure of 8-cell embryos to 40 degrees C enhanced synthesis of 2 constitutive HSP70 proteins (HSC70 and HSC72) and induced another protein, HSP68; exposure of 43 degrees C was required to induce similar responses in expanded blastocysts. Unlike induced thermotolerance, increased synthesis of HSP70 molecules did not depend on whether embryos were cultured or developed in vivo. Thus, other biochemical mechanisms in addition to HSP70 confer thermotolerance in the preimplantation-stage murine embryo. The observation that the temperature threshold for induction of HSP70 synthesis increased from the 8-cell to the blastocyst stage is indicative of these other biochemical processes.     

Gene expression and in vitro development of inter-species nuclear transfer embryos

This study examined the chromatin morphology, in vitro development, and expression of selected genes in cloned embryos produced by transfer of mouse embryonic fibroblasts (MEF) into the bovine ooplasm. After 6 hr of activation, inter-species nuclear transfer (NT) embryos (MEF-NT) had one (70%) or two pronuclei (20%), respectively. After 72 hr of culture in vitro, 62.6% of the MEF-NTs were arrested at the 8-cell stage, 31.2% reached the 2- to 4-cell stage, and only 6.2% had more than eight blastomeres, but none of these developed to the blastocyst stage. Whereas, 20% of NT embryos derived from bovine embryonic fibroblast fused with bovine ooplasm (BEF-NT) reached the blastocyst stage. Donor MEF nuclei expressing an Enhanced Green Fluorescent Protein (EGFP) transgene resulted in 1- to 8-cell stage MEF-NT that expressed EGFP. The expression of selected genes was examined in 8-cell MEF-NTs, 8-cell mouse embryos, enucleated bovine oocytes, and MEFs using RT-PCR. The mRNA for heat shock protein 70.1 (Hsp 70.1) gene was detected in MEF-NTs and MEF, but not in mouse embryos. The hydroxy-phosphoribosyl transferase (HPRT) mRNA was found in normal mouse embryos and MEF but not in MEF-NTs. Expression of Oct-4 and embryonic alkaline phospatase (eAP) genes was only detected in normal mouse embryos and not in the inter-species NT embryos. Abnormal gene expression profiles were associated with an arrest in the development at the 8-cell stage, but MEF-NT embryos appeared to have progressed through gross chromatin remodeling, typical of intra-species NT embryos. Therefore, molecular reprogramming rather than chromatin remodeling may be a better indicator of nuclear reprogramming in inter-species NT embryos.     

Examination of the stress-induced expression of the collagen binding heat shock protein, hsp47, in Xenopus laevis cultured cells and embryos

HSP47 is an endoplasmic reticulum (ER)-resident molecular chaperone involved in collagen production. This study examined the stress-induced pattern of hsp47 gene expression in Xenopus cultured cells and embryos. Sequence analysis revealed that protein encoded by the hsp47 cDNA exhibited 70-77% identity with fish, avian and mammalian HSP47. In A6 kidney epithelial cells hsp47 mRNA and HSP47 were present constitutively and inducible by heat shock but not ER stressors including tunicamycin and A23187, both of which enhanced BiP mRNA. Furthermore A23187 treatment inhibited constitutive accumulation of hsp47 mRNA and retarded heat-induced accumulation of hsp47 and hsp70 mRNA. Interestingly, hsp47 gene expression but not hsp70 or BiP mRNA accumulation was enhanced by treatment with a procollagen-specific stressor, beta-aminopropionitrile. In Xenopus embryos hsp47 mRNA was present constitutively throughout development. In tailbud embryos hsp47 mRNA was enriched in tissues associated with collagen production including notochord, somites and head region. Heat shock-induced accumulation of hsp47 mRNA was enhanced primarily in embryonic tissues already exhibiting hsp47 mRNA accumulation. These studies suggest that the pattern of Xenopus hsp47 gene expression is similar to hsp70 in response to heat shock but also displays unique features including a response to a procollagen-specific stressor and preferential expression in collagen-containing tissues.     

Acquisition of the heat-shock response and thermotolerance during early development of Xenopus laevis

The ability to synthesize a 68,000- to 70,000-Da protein (hsp) in heat-shocked early Xenopus laevis embryos is dependent on the stage of development. Whereas late blastula and later stage embryos synthesize hsp68-70 after heat shock, cleavage stages are incompetent with respect to hsp synthesis. In vitro translation experiments and RNA blot analyses demonstrate that enhanced synthesis of hsp68-70 is associated with an accumulation of hsp68-70 mRNA. Examination of the effect of heat shock on preexisting actin mRNA reveals that heat shock promotes a reduction in the levels of actin mRNA in cleavage embryos but has no discernible effect on actin mRNA levels in neurula embryos. Finally, the acquisition of the heat-shock response (i.e., synthesis of hsp68-70 and accumulation of hsp70 mRNA) during early Xenopus development is correlated with the acquisition of thermotolerance.     

Examination of the expression of the heat shock protein gene, hsp110, in Xenopus laevis cultured cells and embryos

Eukaryotic organisms respond to various stresses with the synthesis of heat shock proteins (HSPs). HSP110 is a large molecular mass HSP that is part of the HSP70/DnaK superfamily. In this study, we have examined, for the first time, the expression of the hsp110 gene in Xenopus laevis cultured cells and embryos. Sequence analysis revealed that the protein encoded by the hsp110 cDNA exhibited 74% identity with its counterparts in mammals and only 27-29% with members of the Xenopus HSP70 family. Hsp110 mRNA and/or protein was detected constitutively in A6 kidney epithelial cells and was inducible by heat shock, sodium arsenite, and cadmium chloride. However, treatment with ethanol or copper sulfate had no detectable effect on hsp110 mRNA levels. Similar results were obtained for hsp70 mRNA except that it was inducible with ethanol. In Xenopus embryos, hsp110 mRNA was present constitutively during development. Heat shock-inducible accumulation of hsp110 mRNA occurred only after the midblastula stage. Whole mount in situ hybridization analysis revealed that hsp110 mRNA accumulation in control and heat shocked embryos was enriched in selected tissues. These studies demonstrate that Xenopus hsp110 gene expression is constitutive and stress inducible in cultured cells and developmentally- and tissue specifically-regulated during early embryogenesis.     

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.     

Translational regulation of the synthesis of a major heat shock protein in HeLa cells

The synthesis of a major heat shock protein (HSP 70) was measured in HeLa cells incubated at 42.5 degrees C and then transferred to 37 degrees C or 30 degrees C. After 90 min, synthesis of HSP 70 decreased by 54 and 85%, respectively, whereas HSP 70 mRNA was reduced at most by 20%. Therefore, the reduced synthesis of HSP 70 could not be accounted for by mRNA turnover. HSP 70 was associated with large polyribosomes (6-10 ribosomes) in cells kept at 42.5 degrees C, but with medium or small polyribosomes in cells transferred to 37 degrees C or 30 degrees C (5-6 or 2-3 ribosomes, respectively). Addition of puromycin to these cells resulted in the release of all ribosomes from HSP 70 mRNA, indicating that they were translationally active. The regulation of HSP 70 synthesis was investigated in cell-free systems prepared from heat-shocked or control cells and incubated at 30 degrees C and 42 degrees C. After 5 min at 42 degrees C, the cell-free system from heat-shocked cells synthesized protein at 3 times the rate of the control cell-free system. This difference was in large part due to synthesis of HSP 70. Addition of HSP mRNA to the control cell-free system stimulated protein synthesis at 42 degrees C, but not at 30 degrees C. These findings suggest that translation of HSP 70 mRNA is specifically promoted at high temperature and repressed during recovery from heat shock by regulatory mechanisms active at the level of initiation.