152. INVOLVEMENT OF TRP53 IN SIRT1 FUNCTION DURING EMBRYO DEVELOPMENT

Abstract

Sirtuin 1 (SIRT1) is an NAD⁺-dependant deacetylase with significant functions in cell survival and metabolism, including glucose homeostasis and mitochondrial physiology. TRP53 is a universal effector of cellular stress responses and is an important target of SIRT1. Transcriptional activity of TRP53 in the pre-implantation embryo is associated with retarded development, however examination of SIRT1 function and how it relates to TRP53 activity remains to be elucidated. We therefore assessed whether SIRT1 is involved in pre-implantation embryo development and determined whether TRP53 interacts with SIRT1 function. Zygotes were collected from superovulated female mice and cultured to the blastocyst stage in optimised conditions (F1 mouse strain, G1/G2 series sequential media, 5%CO₂, 5%O₂, 90%N₂, group culture) or compromised conditions (C57Bl/6 strain, mHTF static media, 5%CO₂ in air, individual culture). Embryo development and blastocyst cell number was assessed following exposure to a SIRT1 inhibitor (0, 1, 10, 100 or 1000µM sirtinol). In subsequent experiments, embryos were cultured in a 2x2 factorial design (±1µM sirtinol and ±30µM pifithrin-α (TRP53 inhibitor)) and embryo development and cell number determined. Sirtinol caused a dose-dependent reduction in total cell number in blastocysts during culture in both optimised and compromised conditions (p<0.05), while the rate of development of zygotes was reduced for embryos in compromised but not optimised conditions (p<0.05). When SIRT1 was inhibited, in the presence or absence of TRP53 inhibition, blastocyst development and cell number for embryos in optimal conditions was unchanged. However, blastocyst development (83% vs 55%, p<0.05), and cell number (39 vs 54 cells, p<0.05) was reduced when SIRT1 was inhibited in compromised embryos, and in the absence of TRP53, development was resolved to control levels. These results show that SIRT1 is important for embryo development, particularly under compromised conditions, and that TRP53 is a likely target for SIRT1 deacetylase activity in the mammalian embryo.