Insulin-like growth factor-I promotes resistance of bovine preimplantation embryos to heat shock through actions independent of its anti-apoptotic actions requiring PI3K signaling

Insulin-like growth factor-I (IGF-I) has been suggested as a survival factor for pre-implantation bovine embryos exposed to heat shock (HS). Therefore, the aims of the study were 1) to examine the protective effects of IGF-I on the developmental competence of bovine oocytes exposed to HS, particularly the effects on oocyte cytoplasmic and nuclear maturation, and 2) to examine whether IGF-I administration contracts HS-induced apoptosis in bovine oocytes. In vitro maturation/IVF/in vitro-production procedures were performed as described previously by Gendelman and Roth (2012). Briefly, cumulus–oocyte complexes (n = 250 to 300/group; 5 replicates) were matured (TCM-199 with Earle’s salts; 22 h, 5% CO2) at 38.5°C or exposed to HS (41°C) with or without 100 µg of IGF-I (Sigma, St. Louis, MO, USA). Matured oocytes were IVF (18 h, 38.5°C, 5% CO2) and cultured in K simplex optimized medium (38.5°C, 5% CO2, 5% O2) for 8 days. Cleavage rates for 2- and 4-cell-stage embryos were assessed at 42 h post-fertilization. For each experimental group, a subgroup of matured oocytes (n = 50) was examined at the end of maturation for nuclear status (1 µg mL–1 of Hoechst 33342, Sigma), cortical granule migration (fluorescein isothiocyanate-Lens culinaris agglutinin, Sigma) and apoptotic status (TUNEL, Roche, Basel, Switzerland). Data were analysed by one-way ANOVA (JMP-6, SAS Institute Inc., Cary, NC, USA) followed by Student’s t-test. Data are presented as mean ± SE. The proportion of oocytes that cleaved to the 2- to 4-cell-stage embryos was lower in the HS group than in the control group (56.55 ± 4.49% v. 75.6 ± 4.16%, respectively; P < 0.05). Although not significant, IGF-I increased the proportions of heat-stressed oocytes that cleaved to the 2- to 4-cell stage (62.32 ± 4.49% v. 56.55 ± 4.49%, for HS + IGF-I and HS, respectively). Neither maturation at 41.5°C nor IGF-I supplementation had any effect on cortical granule migration because the proportions of oocytes with a type I, type II, and type III cortical granule distribution were similar in the control and HS groups. However, the proportion of oocytes that underwent nuclear maturation (i.e. having a nucleus at the telophase-I or metaphase-II stages) was significantly lower in the HS group than in the control group (P < 0.01), and IGF-I slightly increased their proportion in HS oocytes (nonsignificant). The proportion of TUNEL-positive oocytes tended to be higher in the HS group compared with the control group (47.9 ± 12.2% v. 28.0 ± 12.2%, respectively; P ≤ 0.09), and IGF-I decreased the proportion of TUNEL-positive oocytes in the HS group to a level (27.4 ± 12.2%) similar to that noted in the control group. In summary, exposing bovine oocytes to a physiologically relevant thermal stress impaired their ability to undergo first cleavages, most likely because of alteration in nuclear rather than cytoplasmic maturation. Insulin-like growth factor-I was found to slightly alleviate the deleterious effects of heat shock on bovine oocytes.

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

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab110 ◽

2009 ◽

Vol 21 (1) ◽

pp. 155 ◽

Cited By ~ 1

Author(s):

A. Q. Bonilla ◽

P. J. Hansen

Keyword(s):

Growth Factor ◽

Heat Shock ◽

Least Squares ◽

Receptor Expression ◽

Bovine Embryo ◽

Insulin Like Growth Factor ◽

Factor I ◽

Igf I ◽

Rabbit Polyclonal Antibody ◽

Growth Factor I

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.

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