114 Insulin-Like Growth Factor (IGF)2 and IGF2 Receptor (IGF2R) Murine Blastocyst Transcriptional Response after High Gaseous Pressure Exposure at 8-Cell Stage

Abstract

Insulin-like growth factor 2 (IGF2) is a pleiotropic hormone encoded by an imprinted gene expressed in the paternal allele of mammals, and acts in physiological responses including cell proliferation, differentiation, and development. It is mediated through the IGF1R signalling pathway, whereas the IGF2R, a maternally imprinted gene, acts on lysosomal IGF2 degradation. As imprinted genes, both Igf2 and Igf2r expressions are more susceptible to dysregulation by environmental factors. This study aimed to evaluate the effect of exposure of 8-cell-stage murine embryos to 16 MPa of high gaseous pressure (HGP) on the relative Igf2 and Igf2r mRNA abundance in resulting blastocysts following in vitro culture (IVC). Day-3 embryos were recovered from superovulated Mus musculus domesticus females. Eight-cell embryos were exposed to 16 MPa HGP for either 2 h (P1 group) or 4 h (P2 group), with a Control group not exposed to HGP. Immediately after recovery or HGP exposure, embryos were in vitro-cultured for 48 h in mKSOM medium supplemented with 0.4% BSA at 37.5°C, 5% CO₂, 5% O₂, 90% N₂, and saturated humidity. Resulting blastocysts were collected in pools of 10 and stored at –80°C, pending analysis. Following total mRNA extraction, cDNA syntesis and RT-qPCR were performed according to manufacturers. Values were normalized to the internal control Ppia gene. Relative gene expression was calculated using the 2^−ΔΔ Ct approach. Blastocyst rates after IVC were compared by the Chi-squared test (P < 0.05), with relative Igf2 and Igf2r expression data and Igf:Igf2r ratio analysed by ANOVA, after log-transformation when needed, with pairwise comparisons done by the Tukey test (P < 0.05). No differences in blastocyst rates after IVC were observed among groups (Control: 94.2%; P1: 95.4%; P2: 94.1%). However, the Igf2 mRNA relative abundances in blastocysts were 6.3- and 4.2-fold lower in P1 (P < 0.01) and P2 (P = 0.07) than in the Control group, respectively. Likewise, the Igf2r relative transcription levels were 6.6- and 2.2-fold down-regulated in blastocysts from the P1 (P < 0.001) and P2 (P < 0.01) groups, respectively, when compared with controls. Although the relative expression for both genes followed a down-regulation pattern in blastocysts exposed to HGP at the 8-cell stage, the Igf2:Igf2r ratio was 1.9-fold lower in blastocysts in the P2 group (P < 0.05) than Controls, which was similar to the P1 group, indicating a potential stress adaptation response for embryo growth and development after exposure to HGP in the P1 group. It is known that cells under certain conditions of stress may halter growth and development as a response to initiate cellular events to maintain viability. Results from this study appear to translate such response process to HGP in both experimental groups. However, as embryo development and the Igf2:Igf2r ratio in embryos were similar between the Control and the P1 group, exposure to 16 MPa HGP for 2 h at the 8-cell-stage embryo does not seem to affect cell signalling to growth and proliferation up to the blastocyst stage.