110 DEVELOPMENTAL CHANGES IN ACTIONS OF INSULIN-LIKE GROWTH FACTOR-I IN THE PREIMPLANTATION BOVINE EMBRYO-RECEPTOR EXPRESSION AND THERMOTOLERANCE

Abstract

Insulin-like growth factor-I (IGF-I) can affect function of the preimplantation bovine embryo by increasing the proportion of cultured embryos that become blastocysts, reducing effects of heat shock on development and apoptosis, and enhancing survival of embryos transferred into heat-stressed recipients. At Day 5 postinsemination (pi), the embryo is sensitive to IGF-I as determined by activation of the phosphatidylinositol 3-kinase/Akt pathway and activation of thermoprotective mechanisms. It is not known how early in development IGF-I can affect embryo physiology. The overall objective of the present study was to determine whether IGF-I protects two-cell embryos from heat shock. In the first experiment, the presence of the IGF-I receptor (IGF-IR) was evaluated by immunofluorescence using a rabbit polyclonal antibody against a synthetic peptide of the human IGF-IRβ subunit. Specific labeling for IGF-IR was observed for two-cell embryos (n = 20) and embryos ≥16 cells collected at Day 5 pi (n = 17). In the second experiment, it was tested whether IGF-I would protect two-cell embryos from heat shock. Two-cell embryos were collected at 28 hpi and cultured ±100 ng mL^–1 recombinant human IGF-I. After 1 h, embryos were heat-shocked (41°C for 15 h and 38.5°C for 9 h) or maintained at 38.5°C for 24 h. Embryos were then washed to remove IGF-I and cultured in KSOM-BE2 until Day 8 pi. The percent of embryos that became blastocysts at Day 8 was reduced by heat shock (P < 0.005) but was not affected by IGF-I or IGF-I v. heat shock. The least-squares means for percent blastocyst was 38.1% (control) v. 19.3% (heat shock) for embryos without IGF-I and 32.8% (control) v. 20.8% (heat shock) for embryos cultured with IGF-I (n = 11 replicates, n = 169–174 embryos/group; SEM = 2.0%). The third experiment was performed to verify that IGF-I protects Day 5 embryos from heat shock. Embryos ≥16 cells were collected at Day 5 pi and cultured ±100 ng mL^–1 IGF-I. After 1 h, embryos were heat-shocked (42°C for 15 h and 38.5°C for 9 h) or maintained at 38.5°C for 24 h. Embryos were washed and cultured in KSOM-BE2 until Day 8 pi. The percent of embryos that became blastocysts was reduced by heat shock (P < 0.001) and increased by IGF-I (P < 0.05). The least-squares means for percent blastocyst at Day 8 pi was 86.9% (control) v. 47.7% (heat shock) for embryos without IGF-I and 88.7% (control) v. 66.3% (heat shock) for embryos cultured with IGF-I (n = 4 replicates, n = 59–60 embryos/group; SEM = 5.6%). In conclusion, IGF-I does not induce thermotolerance in two-cell embryos despite the presence of IGF-IR.

Support: USDA NRI 2007-35203-18070 and BARD US-3986-07.