125. REGULATION OF STRESS PROTEIN GENES DURING PRE-IMPLANTATION EMBRYO DEVELOPMENT IN MICE BY GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR (GM-CSF)

Abstract

In vitro culture has been shown to be detrimental for pre-implantation embryo development and this has been associated with culture stress and elevated expression of apoptotic genes. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to promote development and survival of both human and mouse pre-implantation embryos. To investigate the mechanism of action of GM-CSF in mouse embryos, gene expression was examined in in vitro cultured blastocysts with and without recombinant mouse GM-CSF (rmGM-CSF) and in vivo blastocysts flushed from Csf2 null mutant and wild-type mice. Microarray analysis of the effect of GM-CSF on transcription profile implicated apoptosis and stress response gene pathways in blastocyst responses to rmGM-CSF in vitro. Groups of 30 blastocysts were collected from in vitro cultured and in vivo developed blastocyst were analysed using quantitative real-time polymerase chain reaction (qRT-PCR). qRT-PCR analysis of in vitro blastocysts revealed that addition of rmGM-CSF causes differential expression of several genes associated with apoptosis and cellular stress pathway, including Cbl, Hspa5, Hsp90aa1, Hsp90ab1 and Gas5. Immunocytochemical analysis of common proteins of the apoptosis and cellular stress response pathways BAX, BCL2, TRP53 (p53) and HSPA1A/1B (Hsp70) in in vitro blastocysts revealed that HSPA1A/1B and BCL2 proteins were less abundant in embryos cultured in rmGM-CSF, but BAX and TRP53 were unchanged. In in vivo developed blastocysts, Csf2 null mutation resulted in elevated levels of only the heat shock protein Hsph1, suggesting that in vivo, other cytokines can compensate for GM-CSF deficiency as the absence of GM-CSF has a lesser effect on the stress response pathway. We conclude that GM-CSF is a regulator of the apoptosis and cellular stress response pathways influencing mouse pre-implantation embryo development to facilitate embryo growth and survival, and the effects of GM-CSF are particularly evident in in vitro culture media in the absence of other cytokines.