85 Regulation of mammalian target of rapamycin signaling post-fertilisation is essential for efficient development of bovine pre-implantation embryos

Abstract

The phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway is considered a key regulator of various cellular responses related to growth, proliferation, survival, apoptosis, and autophagy. The expression of mTOR in cumulus cells and all oocyte stages highlights a role in embryonic development, but its function in the context of embryo development is not completely clarified. In the current study, we investigated the effect of mTOR activation on the total cleavage and blastocyst development rates in bovine. Oocytes collected from bovine ovaries were in vitro matured (around 50 oocytes per group) and fertilised before incubation with the mTOR activator MHY1485 (1 µM) for 72 h at 38.5°C and 5% CO₂. The total cleavage rate at Day 4 post-fertilisation and the blastocyst development rate at Day 8 post-fertilisation were recorded. The expression levels of the mTOR and PI3K in Day 8 blastocysts were investigated using immunofluorescence and imaged using confocal laser-scanning microscope, whereas the fluorescence intensity was analysed using ImageJ software. The difference between groups was analysed using Student's t-test (GraphPad Prism). Results of microscopic investigations showed higher rates of Day 4 cleavage (83.7 ± 1.3 vs. 72 ± 0.8% for control) and Day 8 blastocyst development (40.4 ± 1.7 vs. 33 ± 1.8% for control) upon addition of MHY1485 (P < 0.05). The immunofluorescence showed higher mTOR and PI3K expression levels in MHY1485-treated group than the untreated control (P < 0.05). The total number of cells per blastocyst, using 4′,6-diamidino-2-phenylindole (DAPI) staining of the nuclei, was higher in the mTOR-activated group (194.9 ± 4.8 vs. 171.3 ± 3.5 for control). In conclusion, our data reflect the essential role of the mTOR signaling for bovine pre-implantation embryonic development.