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》

Alterations in ultrastructural morphology of two-cell bovine embryos produced in vitro and in vivo following a physiologically relevant heat shock.

Exposure of cultured preimplantation embryos to temperatures similar to those experienced by heat-stressed cows inhibits subsequent development. In this study, the effects of heat shock on the ultrastructure of two-cell bovine embryos were examined to determine mechanisms for inhibition of development. Two-cell embryos produced in vitro were harvested at approximately 28 h postinsemination and cultured for 6 h at one of three temperatures: 38.5 degrees C (cow body temperature), 41.0 degrees C (characteristic temperature for heat-stressed cows), or 43.0 degrees C (severe heat shock). Ultrastructural examinations revealed that both heat shocks resulted in the movement of organelles towards the center of the blastomere. In addition, heat shock increased the percentage of mitochondria exhibiting a swollen morphology. Distance between the membranes comprising the nuclear envelope was increased but only when embryos were treated at 43.0 degrees C. To determine whether ultrastructural responses to heat shock in culture were similar for embryos produced in vitro and in vivo, two-cell embryos were collected from superovulated Angus cows 48 h postinsemination and treated ex vivo for 6 h at 38.5 degrees C or 41.0 degrees C. Again, heat shock caused an increase in number of swollen mitochondria and movement of organelles away from the periphery of the blastomere. Exposure of two-cell bovine embryos to physiologically relevant elevated temperatures causes disruption in ultrastructural morphology that is inimical to development. The observation that overall morphology and response to heat was similar for embryos produced in vitro and in vivo implies that the former can be a good model for understanding embryonic responses to heat shock.

Rivera RM ，Kelley KL ，Erdos GW ，Hansen PJ ... -  《BIOLOGY OF REPRODUCTION》

Inheritance of resistance of bovine preimplantation embryos to heat shock: relative importance of the maternal versus paternal contribution.

Brahman preimplantation embryos are less affected by exposure to heat shock than Holstein embryos. Two experiments were conducted to test whether the ability of Brahman embryos to resist the deleterious effects of heat shock was a result of the genetic and cellular contributions from the oocyte, spermatozoa, or a combination of both. In the first experiment, Brahman and Holstein oocytes were collected from slaughterhouse ovaries and fertilized with spermatozoa from an Angus bull. A different bull was used for each replicate to eliminate bull effects. On day 4 after fertilization, embryos >or= 9 cells were collected and randomly assigned to control (38.5 degrees C) or heat shock (41 degrees C for 6 hr) treatments. The proportion of embryos developing to the blastocyst (BL) and advanced blastocyst (ABL; expanded and hatched) stages was recorded on day 8. Heat shock reduced the number of embryos produced from Holstein oocytes that developed to BL (P < 0.001, 55.6 +/- 4.2% vs. 29.8 +/- 4.2%) and ABL (P < 0.01, 37.7 +/- 3.6% vs. 12.2 +/- 3.6%) on day 8 as compared to controls. In contrast, heat shock did not reduce development of embryos produced from Brahman oocytes (BL = 42.1 +/- 4.8% vs. 55.6 +/- 4.8% for 38.5 and 41 degrees C, respectively; ABL = 17.6 +/- 4.2% vs. 32.4 +/- 4.2%). In the second experiment, oocytes from Holstein cows were fertilized with semen from bulls of either Brahman or Angus breeds. Heat shock of embryos >or= 9 cells reduced development to BL (P < 0.002) and ABL (P < 0.005) for embryos sired by both Brahman (BL = 54.3 +/- 7.7% vs. 23.4 +/- 7.7%; ABL = 43. +/- 7.4% vs. 7.9 +/- 7.4%, for 38.5 and 41 degrees C, respectively) and Angus bulls (BL = 57.9 +/- 7.7% vs. 31.0 +/- 7.7%; ABL = 33.6 +/- 7.4% vs. 18.4 +/- 7.4%, for 38.5 and 41 degrees C, respectively). There were no breed x temperature interactions. Results suggest that the oocyte plays a more significant role in the resistance of Brahman embryos to the deleterious effects of heat shock than the spermatozoa.

Block J ，Chase CC Jr ，Hansen PJ 《MOLECULAR REPRODUCTION AND DEVELOPMENT》

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 degrees 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 degrees 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 degrees C for the first 12 hr of maturation. Percentages of blastocysts were 35% (39 degrees C), 18% (41 degrees C), 17% (39 degrees C + BSO), and 11% (41 degrees 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 degrees C or 41 degrees C for 12 hr. Exposure of oocytes to 41 degrees 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.

Edwards JL ，Hansen PJ 《MOLECULAR REPRODUCTION AND DEVELOPMENT》

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》