128. A ROLE FOR SIRTUIN 3 IN THE DEVELOPING MAMMALIAN EMBRYO

Abstract

Pre-implantation embryo development relies critically on the balance between cytoplasmic and mitochondrial metabolism for the generation of metabolic intermediates such as NAD⁺_. SIRT3 is a mitochondrial sirtuin with NAD⁺-dependant deacetylase activity that, targets glutamate dehydrogenase (GDH). In this study we characterised SIRT3 mRNA, protein and activity through pre-implantation development and determined whether modulation of SIRT3 activity influenced GDH activity. Embryos (zygotes, 2-cell, 8-cell and blastocyst stages) were recovered from female CBA/C57Bl6 mice following ovarian stimulation and mating with CBA/C57Bl6 males. Expression of SIRT3 mRNA was measured using real-time RTPCR, protein localisation examined using immunohistochemistry and SIRT3 activity measured using a Fluor-de-Lys SIRT3 fluorescent assay. Functional GDH activity was assessed in 2-cell embryos indirectly by measuring glutamine oxidation, following culture from zygote to 2-cell in the presence of nicotinamide, (a sirtuin inhibitor), G1.2 media, or simpleG1 media, compared to in vivo controls. SIRT3 mRNA was detected at all stages of development, with significantly greater levels expressed in the blastocyst. SIRT3 protein was localised predominantly around the nucleus of zygote and 2-cell embryos, and was mainly cytoplasmic in 8-cell embryos and blastocysts. SIRT3 activity remained constant throughout pre-implantation development, and tended to increase at the blastocyst stage. Glutamine oxidation was reduced for embryos cultured in G1.2 media relative to in vivo controls (0.14 pmol/e/hr vs 0.21pmol/e/hr), and this was further reduced by the addition of nicotinamide (0.07pmol/e/hr). Embryo culture in perturbing simpleG1 increased glutamine metabolism (0.33pmol/e/hr). In conclusion, SIRT3 mRNA, protein and activity was detected throughout pre-implantation development. Modulation of sirtuins by nicotinamide decreased glutamine metabolism, likely as a result of decreased deacetylation, thus decreased activity of GDH. SIRT3 can translocate to the mitochondria during cellular stress, thus the increased glutamine metabolism in simpleG1 conditions may be caused by translocation of SIRT3 to mitochondria, potentially increasing GDH deacetylation and enzymatic activity.