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 degrees C), mild heat shock (40 degrees C for 1 h), severe heat shock (42 degrees C for 1 h or 43 degrees 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.

Ealy AD ，Hansen PJ 《JOURNAL OF CELLULAR PHYSIOLOGY》

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.

Al-Katanani YM ，Hansen PJ 《MOLECULAR REPRODUCTION AND DEVELOPMENT》

Development of cultured bovine embryos after exposure to high temperatures in the physiological range.

Embryonic development is inhibited by exposure of cultured embryos to high temperatures. However, culture temperatures used to demonstrate the effects of heat on development have been higher than the body temperatures experienced typically by heat-stressed cows. The aim of this study was to determine whether exposing bovine oocytes and embryos to temperatures characteristic of body temperatures of heat-stressed cows would affect embryonic development in vitro. The CO2 percentage of the gas phase was adjusted in all experiments to prevent pH changes in the medium caused by decreased solubility of CO2 at high temperatures. Fertilization of oocytes at 41.0 degrees C reduced cleavage rate and the percentage of oocytes that became blastocysts compared with at 38.5 degrees C. There was no deleterious effect of fertilization at 40.0 degrees C. When putative zygotes and two-cell embryos were exposed to a range of temperatures from 38.5 to 41.0 degrees C for 3, 6, 9 or 12 h, heat shock reduced the number that developed to the blastocyst stage but only after exposure to 41.0 degrees C for 9 or 12 h. In addition, it was tested whether low O2 tension would reduce the detrimental effects of heat shock. The deleterious effect of 41.0 degrees C was not dependent upon oxygen content or the gas mixture used for culture (5% versus 20.95% O2), indicating that the deleterious effects of heat shock did not depend upon a high O2 environment. In the final experiment, embryos were exposed to 24 h fluctuations in temperature designed to mimic the rectal temperatures of cows exposed to heat stress. Exposure of embryos to this pattern of temperatures starting after fertilization reduced development when embryos were exposed to this environment for 8 days but not when embryos were exposed for 1 day only. These findings indicate that embryonic development can be disrupted by a short-term severe or a prolonged mild heat shock and that the effects of heat shock are not artefacts of changes in pH or high oxygen tension.

Rivera RM ，Hansen PJ 《REPRODUCTION》

Thermotolerance of IVM-derived bovine oocytes and embryos after short-term heat shock.

A series of experiments were designed to study the effect of elevated temperatures on developmental competence of bovine oocytes and embryos produced in vitro. In experiment 1, the effect of heat shock (HS) by a mild elevated temperature (40.5 degrees C) for 0, 30, or 60 min on the viability of in vitro matured (IVM) oocytes was tested following in vitro fertilization (IVF) and culture. No significant difference was observed between the control (39 degrees C) and the heat-treated groups in cleavage, blastocyst formation, or hatching (P > 0.05). In experiment 2, when the HS temperature was increased to 41.5 degrees C, neither the cleavage rate nor blastocyst development was affected by treatment. However, the rate of blastocyst hatching appeared lower in the HS groups (13% in control group vs. 3.9% and 5.6% in 30 min and 60 min, respectively; P < 0.05). When IVM oocytes were treated at 43 degrees C prior to IVF (experiment 3), no difference was detected in blastocyst and expanded blastocyst development following heat treatment for 0, 15, or 30 min, but heat treatment of oocytes for 45 or 60 min significantly reduced blastocyst and expanded blastocyst formation (P < 0.05). In experiment 4, the thermotolerance of day 3 and day 4 bovine IVF embryos were compared. When embryos were pre-treated with a mild elevated temperature (40.5 degrees C) for 1 hr, and then with a higher temperature (43 degrees C) for 1 hr, no improvement in thermotolerance of the embryos was observed as compared to those treated at 43 degrees C alone. However, a higher thermotolerance was observed in day 4 than day 3 embryos. In conclusion, treatment at 43 degrees C, but not 40.5 degrees C or 41.5 degrees C significantly reduced oocyte developmental competence. An increase in thermotolerance was observed from day 3 to day 4 of in vitro embryonic development, which corresponds to the maternal to zygotic transition of gene expression in bovine embryos.

Ju JC ，Parks JE ，Yang X 《MOLECULAR REPRODUCTION AND DEVELOPMENT》

Ontogeny of temperature-regulated heat shock protein 70 synthesis in preimplantation bovine embryos.

Development of the preimplantation embryo is very susceptible to disruption by heat shock. As embryos proceed through development, they acquire resistance to heat shock, perhaps because they become transcriptionally active and can respond to environmental changes by undergoing transcriptionally-regulated cellular adaptation. Objectives were to determine the ontogeny of heat shock protein 70 (HSP70) synthesis in preimplantation bovine embryos and to ascertain whether heat-induced increases in HSP70 in embryos are caused by environmental alterations in gene expression. Exposure of bovine embryos to heat shock induced synthesis of a 68 kDa form of HSP70 called HSP68 as early as the two-cell stage of development. Induction of HSP68 was alpha-amanitin independent at the two-cell stage but was blocked by alpha-amanitin as early as the early four-cell stage. Therefore, heat-induced synthesis of HSP68 is regulated at the level of transcription at a time before the major round of embryonic genome activation is considered to occur. Two other constitutive HSP70 molecules were identified called heat shock cognates (HSC) 71 and 70; both proteins were synthesized during all stages of development from the two-cell to hatched blastocyst stages. However, heat-induced synthesis of HSC71 and HSC70 was not evident until the expanded blastocyst stage. In conclusion, environmental signals can activate gene expression before the major round of embryonic genome activation occurs in bovine embryos. Moreover, differences in thermal sensitivity of early embryos to heat shock is not caused by an inability to synthesize HSP70, suggesting that other mechanisms are involved in developmental acquisition of thermotolerance.

Edwards JL ，Ealy AD ，Monterroso VH ，Hansen PJ ... -  《MOLECULAR REPRODUCTION AND DEVELOPMENT》