Heat Shock-Induced Apoptosis in Preimplantation Bovine Embryos: MATERIALS AND METHODS(5)

Experiments

A series of six experiments was conducted to evaluate heat-induced apoptosis. For TUNEL analysis, embryos were collected at different times in development, transferred to a new drop of modified KSOM (15-30 embryos per drop; within a run, similar numbers of embryos per drop were used for each treatment), and maintained at 38.5°C for 24 h or exposed to various heat shock (40, 41, or 42°C) of 3- to 12-h duration. A heat shock of 42°C was used in the initial experiment to maximize probability of induction of apoptosis. In subsequent experiments, embryos were exposed to more physiological temperatures (40-41°C) that were similar to those experienced by heat-stressed cows. Heat-shocked embryos were then returned to 38.5°C. At 24 h after the initiation of heat shock, embryos were fixed in 4% (w/v) paraformaldehyde, transferred to a poly-L-lysine coated slide and saved at 4°C until analysis by TUNEL. For caspase activity, embryos were exposed to either 38.5°C or 41°C for 9 h. Immediately after the end of heat shock, caspase activity was determined as previously described. Details of specific experiments are described below.

Heat-induced apoptosis in bovine embryos a 16-cell stage. Bovine embryos that were a 16 cells were collected on Day 5 after insemination and transferred to a new drop of modified KSOM. Embryos were cultured at 38.5°C for 24 h or were heat shocked at 41 or 42°C for 9 h followed by 38.5°C for 15 h. Analysis by TUNEL was then performed. The experiment was replicated four times using 208 embryos (50-85 embryos/ group).

Effect of magnitude and duration of heat shock on heat-induced apoptosis. The purpose of this experiment was to determine whether the degree of apoptosis was proportional to the severity of heat shock. This was accomplished by heat shocking embryos at two different temperatures for variable durations. Embryos a 16 cells were collected on Day 5 after insemination and transferred to a new drop of modified KSOM (15-30 embryos per drop). Embryos were cultured at either 38.5°C for 24 h or were heat shocked at 40 or 41°C for 3, 6, or 9 h followed by 38.5°C for a total culture period of 24 h. Twenty-four hours after the initiation of heat shock, embryos were fixed in 4% paraformaldehyde, transferred to a poly-L-lysine coated slide, and saved at 4°C until analysis by TUNEL. The experiment was replicated four times using 342 embryos (30-70 embryos/ group).

Category: Embryo

Tags: Apoptosis, Early development, Embryo, Stress

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