121 DEVELOPMENTAL CHANGES IN THERMOPROTECTIVE ACTIONS OF INSULIN-LIKE GROWTH FACTOR-1 ON THE PREIMPLANTATION BOVINE EMBRYO

Abstract

Insulin-like growth factor-1 (IGF1) is an important endocrine signal for regulation of early embryonic development. It increases the proportion of preimplantation embryos becoming blastocysts, alters blastocyst gene expression, improves resistance of embryos to various stresses and can enhance survival of embryos after transfer to recipients. The present study had 2 objectives. The first was to determine whether the thermoprotective actions of IGF1 on the preimplantation bovine embryo were developmentally regulated, with the 2-cell embryo being refractory to IGF1. The second was to determine the molecular basis for the improved competence of embryos treated with IGF1 to establish pregnancy after transfer to heat-stressed recipients. Heat shock at 41°C decreased (P < 0.005) the percentage of 2-cell embryos becoming a blastocyst at day 8 (39.5 v. 21% for 38.5 and 41°C, respectively), and treatment of embryos with 100 ng mL^–1 IGF1 did not provide thermoprotection to 2-cell embryos heat shocked at 41°C (21 v. 21% for control and IGF1-treated embryos, respectively). Heat shock at 41°C had no effect on blastocyst development of day 5 embryos. However, exposure to 42°C reduced (P < 0.001) blastocyst development of day 5 embryos (87 v. 47.6% for 38.5 and 42°C, respectively). Furthermore, treatment of embryos with 100 ng mL^–1 IGF1 reduced (P = 0.05) the effect of heat shock at 42°C on day 5 embryos (48 v. 66% control and IGF1-treated embryos, respectively). Failure of IGF1 to alter 2-cell embryo survival after heat shock was not associated with reduced expression of genes involved in IGF1 signaling (IGF1R, RAF1, PI3K, and MAPK), as shown by quantitative real-time RT-PCR assay, or in amounts of immunoreactive IGF1R protein. Treatment with IGF1 had little effect on the transcriptome at the blastocyst stage, with a total of 102 differentially expressed genes identified. Among the differentially expressed genes were several involved in apoptosis, protection against free radicals, and development. Changes in gene expression are consistent with IGF1 acting to induce an anti-apoptotic state and inhibit neurulation. In conclusion, thermoprotective actions of IGF1 are developmentally regulated. Failure of IGF1 to protect the 2-cell embryo from heat shock could reflect the fact that these embryos are maximally sensitive to damage caused by heat shock or reflect the quiescence of the embryonic genome at this early stage in development. Changes in gene expression at the blastocyst stage induced by IGF1 could contribute to the increased survival of IGF1-treated embryos when transferred during periods of heat stress.