129 INSULIN-LIKE GROWTH FACTOR-I PROMOTES BLASTOCYST DEVELOPMENT OF HEAT-SHOCKED BOVINE EMBRYOS INDEPENDENT OF ITS ANTI-APOPTOTIC EFFECTS REQUIRING P13K SIGNALING

Abstract

Insulin-like growth factor-I (IGF-I) reduces effects of heat shock on blastocyst development and induction of apoptosis. The present objective was to test whether IGF-I would allow for blastocyst development following heat shock because of its anti-apoptotic effects. Because anti-apoptotic actions of IGF-I require signaling through the phosphatidylinositol 3-kinase (PI3K) pathway, an inhibitor of PI3K (LY 294002) was used in Exp. 1 to determine whether it would prevent the thermoprotective effects of IGF-I on development. Embryos were produced in vitro in KSOM-BE2 medium ±100 ng mL^-1 of IGF-I. Embryos e16 cells at 5 d post-insemination (dpi) were placed in fresh drops containing the same IGF-I treatment as well as LY 294002 (100 ¼m) or vehicle (0.1% DMSO) and cultured at either 38.5°C or 41°C for 15 h. All groups were then cultured at 38.5°C until 8 dpi when blastocyst development was assessed (10 replicates; 112 to 142 embryos per treatment). For embryos in DMSO, IGF-I did not increase the percentage that became blastocysts when cultured at 38.5°C (59.1 and 57.1% for control and IGF-I embryos, respectively; SEM = 3.6%). Heat shock reduced blastocyst development (P < 0.01), and IGF-I blocked this decrease (percent blastocyst = 42.0 and 54.7% for control and IGF-I embryos, respectively; SEM = 3.6%). Similar results were obtained for embryos cultured with LY 294002. The percentage of embryos becoming blastocysts for control embryos was 53.5 and 35.5%, respectively, at 38.5 and 41°C, development for IGF-I-treated embryos was, respectively, 55.8 and 49.4% at 38.5 and 41°C (SEM = 4.8%). Analysis of the entire data set revealed an IGF-I × temperature interaction (P < 0.05) but no interactions with inhibitor treatment. Thus, IGF-I protected embryos from heat shock in the presence and absence of LY 294002. In Exp. 2, procedures were similar, except that embryos were cultured with a caspase-3 inhibitor (z-DEVD-fmk; 100 ¼m) instead of LY 294002 (12 replicates; 114 to 137 embryos per treatment). For embryos in DMSO, blastocyst development was reduced by heat shock (P < 0.06) and increased by IGF-I (P < 0.06). The percentage of embryos becoming blastocysts was 67.0 and 55.8% for control embryos at 38.5 and 41°C, respectively, vs. 74.7 and 70.8% for IGF-I embryos at 38.5 and 41°C, respectively (SEM = 5.2%). For embryos cultured with z-DEVD-fmk, heat shock reduced blastocyst development (P < 0.01), and IGF-I was no longer effective in blocking the reduction in blastocyst development caused by heat shock (IGF-I × inhibitor treatment; P < 0.01). Blastocyst development was 66.4 and 58.7% for control embryos at 38.5 and 41°C, respectively, vs. 61.8 and 48.6% for IGF-I embryos at 38.5 and 41°C, respectively (SEM = 4.6%). Note that z-DEVD-fmk did not protect embryos from the anti-developmental effects of heat shock but exacerbated heat shock effects for IGF-I-treated embryos. In conclusion, the ability of IGF-I to allow heat-shocked embryos to continue development to the blastocyst stage is independent of its anti-apoptotic effects involving the PI3K pathway but may depend on active caspase-3.

This work was supported by USDA NRICGP 2002-35203-12664, BARD US-3551-04, and USDA TSTAR 2004-34135-14715.