(361) Effect of Bovine In Vitro Embryo Production on Fetal IGF System Signaling

Hector Nava-Trujillo¹; Christine Spinka²; Rocio Melissa Rivera¹

¹ Division of Animal Sciences, University of Missouri, Columbia, MO, USA

² Department of Biomedical Informatics, Biostatistics, and Medical Epidemiology, School of Medicine, University of Missouri, Columbia, MO, USA

Abstract Text:

In vitro production (IVP) of embryos is used to promote rapid genetic improvement in cattle.  Currently, ~1.6 million embryos are produced worldwide yearly for this purpose. Despite the measurable genetic improvement of desirable traits, and similar pregnancy rates compared to artificial insemination (AI), IVP embryos exhibit epigenetic alterations affecting the expression of imprinted and non-imprinted genes, resulting in altered fetal and placental growth. Genomic imprinting is an epigenetic mechanism controlling parental allele-specific expression of genes that regulate growth and development of the fetus and the placenta. We have shown that IVP-induced fetal overgrowth is correlated to altered methylation of various imprinted domains.  Results from our and other laboratories have identified insulin-like growth factor receptor 2 (IGF2R), a maternally-expressed gene, to be downregulated in overgrowth.  IGF2R targets IGF2 (a paternally-expressed fetal growth factor) for degradation. IGF2 signals via the IGF1R and insulin receptor A to stimulate the PI3K-AKT pathway.  Here we aimed to establish if IVP induces alterations in the IGF signaling system in day 105 conceptuses. In an embryo transfer experiment, the variation in the weight was higher in IVP fetuses (n=42) than in AI controls (n=11) at day 105 of gestation (25.94% vs. 11.93%, respectively, p=0.0214).  The transcript amount of IGF2R in the muscle and placenta (i.e. cotyledon) were negatively correlated with fetal weight only in IVP fetuses (p< 0.0001).  We used a subset of fetuses in the AI (n=8) and IVP (n=8) groups to start characterizing the IGF signaling pathways in muscle and liver (placenta analyses are underway).  Liver is the main production site of IGF2 in the conceptus which is released into the bloodstream to affect target tissues, including muscle. For this, the abundance of IGF2, IGF1R, IGF2R and of proteins in the PI3K/AKT pathway (PI3K subunit p110 total and phosphorylated, AKT total and phosphorylated and mTOR total and phosphorylated) were detected with Western blot and protein-specific results were normalized using total protein normalization method and quantified using ImageJ (NIH). Data were analyzed by ANOVA and the group means were compared by least squares (the model was corrected for sex).  Initial results show that liver of fetuses derived from IVP had an increase of 477% in the abundance of IGF2 (p=0.03) and this was accompanied with a reduction of 18% in the abundance of IGF2R (p=0.07), while no differences were observed in the abundance of IGF1R. In muscle, and contrary to liver, IVP fetuses had a reduction of 53.12% of IGF2 (p< 0.0001) and an increase of 54.28% in the abundance of IGF1R (p=0.0021), while no differences were observed for IGF2R.  Next, we examined abundance of kinases of the PI3K/AKT pathway in fetal muscle and found that that IVP fetuses had 47.8% less p110 total than controls (p=0.001) but an 81.3% increase in the ratio of phosphorylated p110 (p=0.001), indicating an increased rate of activation of this kinase. No differences were observed in the abundance of AKT total and phosphorylated between groups. The total levels of mTOR was increased by 58.45% (p=0.08) and phosphorylated mTOR was decreased by 45.37% (p=0.01) in IVP derived fetuses. The ratio of phosphorylated mTOR was reduced 68.44% in muscle of IVP fetuses (p=0.01). In summary, these preliminary results suggest that epigenetic alterations occur in IVP embryos prior to transfer which affect regulation of the IGF signaling system during fetal development in cattle.