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.

Krininger CE 3rd ，Stephens SH ，Hansen PJ 《MOLECULAR REPRODUCTION AND DEVELOPMENT》

Insulin-like growth factor-I as a survival factor for the bovine preimplantation embryo exposed to heat shock.

Insulin-like growth factor-I (IGF-I) is a survival factor for preimplantation mammalian embryos exposed to stress. One stress that compromises preimplantation embryonic development is elevated temperature (i.e., heat shock). Using bovine embryos produced in vitro as a model, it was hypothesized that IGF-I would protect preimplantation embryos by reducing the effects of heat shock on total cell number, the proportion of blastomeres that undergo apoptosis, and the percentage of embryos developing to the blastocyst stage. In experiment 1, embryos were cultured with or without IGF-I; on Day 5 after insemination, embryos >or=16 cells were cultured at 38.5 degrees C for 24 h or were subjected to 41 degrees C for 9 h followed by 38.5 degrees C for 15 h. Heat shock reduced the total cell number at 24 h after initiation of heat shock and increased the percentage of blastomeres that were apoptotic. Effects of heat shock were less for IGF-I-treated embryos. Experiment 2 was conducted similarly except that embryos were allowed to develop to Day 8 after insemination. The percentage reduction in blastocyst development for heat-shocked embryos compared with those maintained at 38.5 degrees C was less for embryos cultured with IGF-I than for control embryos. Heat shock reduced the total cell number in blastocysts and increased the percentage of blastomeres that were apoptotic, whereas IGF-I-treated embryos had increased total cell number and a reduced percentage of apoptosis. Taken together, these results demonstrate that IGF-I can serve as a survival factor for preimplantation bovine embryos exposed to heat shock by reducing the effects of heat shock on development and apoptosis.

Jousan FD ，Hansen PJ 《BIOLOGY OF REPRODUCTION》

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》

To be or not to be--determinants of embryonic survival following heat shock.

Elevated temperature can reduce developmental competence of the preimplantation embryo. Whether an embryo survives elevated temperature depends on its genotype, stage of development, exposure to regulatory molecules and redox status. Following fertilization, the embryo is very sensitive to heat shock. By Days 4-5 after insemination, however, the embryo has acquired increased resistance to elevated temperature. One system that may potentiate embryonic survival at later stages of embryonic development is the apoptosis response-inhibition of apoptosis responses at Day 4 exacerbated effects of heat shock on development. Embryo responses to heat shock at Days 4-5 also depend upon genotype because Bos indicus embryos are more resistant than embryos from non-adapted B. taurus. Some experiments (although not all) indicate that survival following heat shock can be increased by reducing oxygen tension, suggesting involvement of reactive oxygen species or hypoxia-induced factors. Embryonic responses to heat shock are also affected by regulatory molecules that act to modify cellular physiology and improve cell survival. The best characterized of these is insulin-like growth factor-1 (IGF-1). Actions of IGF-1 to allow development following heat shock are independent of its anti-apoptotic actions because inhibition of the phosphatidylinositol-3 kinase pathway through which IGF-1 blocks apoptosis does not prevent thermoprotective effects of IGF-1 on development. Identification of specific determinants of embryonic survival creates the opportunity for new strategies to improve pregnancy rates in animals exposed to heat stress. Many environmental perturbations activate similar cellular responses. Therefore, molecular and cellular systems that improve embryonic survival to heat shock may confer protection from other embryotoxic conditions.

Hansen PJ 《THERIOGENOLOGY》

Ceramide inhibits development and cytokinesis and induces apoptosis in preimplantation bovine embryos.

de Castro e Paula LA ，Hansen PJ 《-》